Bispecific antibody for icos and pd-l1

ABSTRACT

Multispecific antibody in mAb2 format, comprising an ICOS-binding Fab region and a PD-L1-binding Fcab region. Use of the multispecific antibody in immuno-oncology, including for treatment of solid tumours. Combination therapy including antibody to another immune checkpoint molecule such as PD-1 and CTLA-4, in addition to anti-ICOS and anti-PD-L1.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/955,219, filed Jun. 18, 2020, which is a 35 U.S.C. § 371 filing of International Patent Application No. PCT/GB2018/053698, filed Dec. 19, 2018, which claims priority to U.S. Provisional Patent Application Ser. No. 62/607,469, filed Dec. 19, 2017, the entire disclosures of which are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created Jul. 20, 2023, is named 740018_SA9-621USCON_ST26.xml and is 670,276 bytes in size.

FIELD OF THE INVENTION

This invention relates to antigen-binding molecules that bind cell surface receptors involved in regulation of the immune response. It relates to antibodies for use in stimulating a patient's immune system, especially the effector T cell response, and has applications in the field of immuno-oncology, especially treatment of tumours. More particularly the invention relates to multi-specific antibodies that bind ICOS and PD-L1.

BACKGROUND

An adaptive immune response involves activation, selection, and clonal proliferation of two major classes of lymphocytes termed T cells and B cells. After encountering an antigen, T cells proliferate and differentiate into antigen-specific effector cells, while B-cells proliferate and differentiate into antibody-secreting cells. T cell activation is a multi-step process requiring several signalling events between the T cell and an antigen-presenting cell (APC). For T cell activation to occur, two types of signals must be delivered to a resting T cell. The first type is mediated by the antigen-specific T cell receptor (TCR), and confers specificity to the immune response. The second signal, a costimulatory signal, regulates the magnitude of the response and is delivered through accessory receptors on the T cell.

A primary costimulatory signal is delivered through the activating CD28 receptor upon engagement of its ligands B7-1 or B7-2. In contrast, engagement of the inhibitory CTLA-4 receptor by the same B7-1 or B7-2 ligands results in attenuation of a T cell response. Thus, CTLA-4 signals antagonize costimulation mediated by CD28. At high antigen concentrations, CD28 costimulation overrides the CTLA-4 inhibitory effect. Temporal regulation of the CD28 and CTLA-4 expression maintains a balance between activating and inhibitory signals and ensures the development of an effective immune response, while safeguarding against the development of autoimmunity.

Programmed death-1 (PD-1) is a 50-55 kDa type I transmembrane receptor that is a member of the CD28 family. PD-1 is involved in the regulation of T-cell activation and is expressed on T cells, B cells, and myeloid cells. Two ligands for PD-1, PD ligand 1 (PD-L1) and ligand 2 (PD-L2) have been identified and have costimulatory features.

Programmed cell death 1 ligand 1 (PD-L1), also known as cluster of differentiation (CD274) or B7 homolog 1 (B7-H1), is a member of the B7 family that modulates activation or inhibition of the PD-1 receptor. The open reading frame of PD-L1 encodes a putative type 1 transmembrane protein of 290 amino acids, which includes two extracellular Ig domains (an N-terminal V-like domain and an Ig C-like domain), a hydrophobic transmembrane domain and a cytoplasmic tail of 30 amino acids. The 30 amino acid intracellular (cytoplasmic) domain contains no obvious signalling motifs, but does have a potential site for protein kinase C phosphorylation. The complete amino acid sequence for PD-L1 can be found in NCBI Reference Sequence: NP_054862.1 (SEQ ID NO: 1), which refers to many journal articles [1]. The PD-L1 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, and zebrafish. The murine form of PD-L1 bears 69% amino acid identity with the human form of PD-L1, and also shares a conserved structure.

In humans, PD-L1 is expressed on a number of immune cell types including activated and anergic/exhausted T cells, on naive and activated B cells, as well as on myeloid dendritic cells (DC), monocytes and mast cells. It is also expressed on non-immune cells including islets of the pancreas, Kupffer cells of the liver, vascular endothelium and selected epithelia, for example airway epithelia and renal tubule epithelia, where its expression is enhanced during inflammatory episodes. PD-L1 expression is also found at increased levels on a number of tumours, such as breast (e.g., triple negative breast cancer and inflammatory breast cancer), ovarian, cervical, colon, colorectal, lung (e.g., non-small cell lung cancer), renal (e.g., renal cell carcinoma), gastric, oesophageal, bladder, hepatocellular cancer, squamous cell carcinoma of the head and neck (SCCHN) and pancreatic cancer, melanoma and uveal melanoma.

PD-1/PD-L1 signalling is believed to serve a critical non-redundant function within the immune system by negatively regulating T cell responses. This regulation is involved in T cell development in the thymus, in regulation of chronic inflammatory responses and in maintenance of both peripheral tolerance and immune privilege. It appears that upregulation of PD-L1 may allow cancers to evade the host immune system and, in many cancers, the expression of PD-L1 is associated with reduced survival and an unfavourable prognosis. Therapeutic monoclonal antibodies that are able to block the PD-1/PD-L1 pathway may enhance anti-tumoural immune responses in patients with cancer. Published clinical data suggest a correlation between clinical responses with tumoural membranous expression of PD-L1 and a stronger correlation between lack of clinical responses and a lack of PD-L1 protein localised to the membrane [2, 3]. Thus, PD-L1 expression in tumours or tumour-infiltrating leukocytes is a candidate molecular marker for use in selecting patients for immunotherapy, for example, immunotherapy using anti-PD-L1 antibodies [4]. Patient enrichment based on surface expression of PD-L1 may significantly enhance the clinical success of treatment with drugs targeting the PD-1/PD-L1 pathway. There is also evidence of an ongoing immune response, such as the tumour infiltrating CD8+ T cells, or the presence of signature of cytokine activation, such as IFNγ.

Further evidence of PD-L1 expression and correlation to disease will emerge from the numerous ongoing clinical trials. Atezolizumab is the most advanced anti-PD-L1 antibody in development, and Phase II trials showed therapeutic effects in metastatic urothelial carcinoma and NSCLC, particularly in patients with PD-L1⁺ immune cells in the tumour microenvironment [5, 6]). Recent results from a Phase III trial of 1225 patients with NSCLC showed improved survival in patients taking atezolizumab, compared with chemotherapy, regardless of tumour expression of PD-L1 (Rittmeyer et al., 2017, The Lancet, 389(10066), 255-265).

Another member of the CD28 gene family, ICOS (Inducible T cell Co-Stimulator), was identified in 1999 [7]. It is a 55 kDa transmembrane protein, existing as a disulphide linked homodimer with two differentially glycosylated subunits. ICOS is exclusively expressed on T lymphocytes, and is found on a variety of T cell subsets. It is present at low levels on naïve T lymphocytes but its expression is rapidly induced upon immune activation, being upregulated in response to pro-inflammatory stimuli such as on engagement of TCR and co-stimulation with CD28 [8, 9]. ICOS plays a role in the late phase of T cell activation, memory T cell formation and importantly in the regulation of humoral responses through T cell dependent B cell responses [10, 11]. Intracellularly, ICOS binds PI3K and activates the kinases phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB). Activation of ICOS prevents cell death and upregulates cellular metabolism. In the absence of ICOS (ICOS knock-out) or in the presence of anti-ICOS neutralising antibodies there would be a suppression of pro-inflammatory responses.

ICOS binds to ICOS ligand (ICOSL) expressed on B-cells and antigen presenting cells (APC) [12, 13]. As a co-stimulatory molecule it serves to regulate TCR mediated immune responses and antibody responses to antigen. The expression of ICOS on T regulatory cells may be important, as it has been suggested that this cell type plays a negative role in immunosurveillance of cancer cells—there is emerging evidence for this in ovarian cancer [14]. Importantly, ICOS expression has been reported to be higher on intratumoural regulatory T cells (TRegs) compared with CD4+ and CD8+ effector cells that are present in the tumour microenvironment. Depletion of TRegs using antibodies with Fc-mediated cellular effector function has demonstrated strong anti-tumour efficacy in a pre-clinical model [15]. Mounting evidence implicates ICOS in an anti-tumour effect in both animal models as well as patients treated with immune-checkpoint inhibitors. In mice deficient in ICOS or ICOSL the anti-tumor effect of anti-CTLA4 therapy is diminished [16] while in normal mice ICOS ligand increases the effectiveness of anti-CTLA4 treatment in melanoma and prostate cancer [17]. Furthermore, in humans a retrospective study of advanced melanoma patients showed increased levels of ICOS following ipilimumab (anti-CTLA4) treatment [18]. In addition, ICOS expression is upregulated in bladder cancer patients treated with anti-CTLA4 [19]. It has also been observed that in cancer patients treated with anti-CTLA4 therapy the bulk of tumour specific IFNγ producing CD4 T-cells are ICOS positive while sustained elevation of ICOS positive CD4 T cells correlates with survival [18, 19, 20].

WO2016/120789 described anti-ICOS antibodies and proposed their use for activating T cells and for treating cancer, infectious disease and/or sepsis. A number of murine anti-ICOS antibodies were generated, of which a sub-set were reported to be agonists of the human ICOS receptor. The antibody “422.2” was selected as the lead anti-ICOS antibody and was humanised to produce a human “IgG4PE” antibody designated “H2L5”. H2L5 was reported to have an affinity of 1.34 nM for human ICOS and 0.95 nM for cynomolgus ICOS, to induce cytokine production in T cells, and to upregulate T cell activation markers in conjunction with CD3 stimulation. However, mice bearing implanted human melanoma cells were reported to show only minimal tumour growth delay or increase in survival when treated with H2L5 hIgG4PE, compared with control treated group. The antibody also failed to produce significant further inhibition of tumour growth in combination experiments with ipilimumab (anti-CTLA-4) or pembrolizumab (anti-PD-1), compared with ipilimumab or pembrolizumab monotherapy. Finally, in mice bearing implanted colon cancer cells (CT26), low doses of a mouse cross reactive surrogate of H2L5 in combination with a mouse surrogate of ipilimumab or pembrolizumab only mildly improved overall survival compared with anti-CTL4 and anti-PD1 therapy alone. A similar lack of strong therapeutic benefit was shown in mice bearing implanted EMT6 cells.

WO2016/154177 described further examples of anti-ICOS antibodies. These antibodies were reported to be agonists of CD4+ T cells, including effector CD8+ T cells (TEff), and to deplete T regulator cells (TRegs). Selective effects of the antibodies on TEff vs TReg cells were described, whereby the antibodies could preferentially deplete TRegs while having minimal effect on TEffs that express a lower level of ICOS. The anti-ICOS antibodies were proposed for use in treating cancer, and combination therapy with anti-PD-1 or anti-PD-L1 antibodies was described.

Although there has been immense progress in the field of immuno-oncology in recent years, current response rates of immuno-oncology drugs remain low. For example, the response rate for the anti-PD-1 antibody nivolumab in melanoma is around 30%, and the response rate for the anti-PD-L1 atezolizumab in its Phase II clinical trial in urothelial carcinoma was around 15% overall in patients regardless of PD-L1 expression or 26% in patients with PD-L1 expressing tumours. Efforts to increase efficacy of immuno-oncology treatment have included combining multiple drugs, for example combinations of antibodies and traditional chemotherapeutic agents or radiation, and the combined use of drugs targeting different immune checkpoint inhibitors. A combination of nivolumab (anti-PD-1) and ipilimumab (anti-CTLA-4) has shown efficacy in previously untreated cases of melanoma, with headline response rates and overall survival being encouraging [21]. However, although combination therapy may generate new or enhanced biological effects in vivo, this carries an associated risk of negative drug interactions and new or worsened side-effects. Immune checkpoint inhibitor therapy is already associated with immune-related adverse events, including neurological events ranging from mild headache to life-threatening encephalitis [22]. Further, on a practical level, treatment regimens involving combinations of multiple therapeutic agents have the drawbacks of complex administration regimens and high cost.

SUMMARY OF THE INVENTION

The present invention relates to antigen-binding molecules that comprise multiple antigen-binding sites (“multispecific antigen-binding molecules”), including an antigen-binding site for human ICOS and an antigen-binding site for human PD-L1. The multispecific antibodies of the present invention are in the format known as “mAb2” or “mAb squared”, which are antibodies comprising an Fc region that has been engineered to contain antigen-binding loops in its CH3 domain—this modified Fc is termed an “Fcab”. The mAb2 further comprises a Fab region, comprising a VH-VL domain pair providing an antigen-binding site. mAb2 molecules of the present invention comprise a PD-L1 binding Fcab and an ICOS-binding Fab. See FIG. 3 .

Accordingly, a first aspect of the present invention is a multispecific antibody comprising a Fab and an Fc region, wherein the Fab comprises an ICOS binding site and wherein the Fc region comprises a PD-L1 binding site. The PD-L1 binding site is engineered into loop regions of the CH3 domain of the Fc region. The Fc comprises a CH3 domain in which residues 14 to 18 (the “AB loop”) are substituted by SGYW (SEQ ID NO: 617), residues 45.1 to 78 (the “CD loop”) are substituted by EPQYWA (SEQ ID NO: 618), and residues 92 to 101 (the “EF loop”) are substituted by SNWRWQLDD (SEQ ID NO: 619). Numbering is according to IMGT. See FIG. 2 , which shows the CH3 domain sequence of the Fcab aligned against a wild type human IgG1 sequence, with IMGT numbering. Thus, the multispecific antibody of the present invention comprises an Fcab CH3 domain comprising PD-L1 binding site provided by structural loop sequences as follows:

-   -   AB loop SEQ ID NO: 617     -   CD loop SEQ ID NO: 618     -   EF loop SEQ ID NO: 619.

An Fc region is dimeric and thus comprises paired heavy chain constant regions each comprising a CH3 domain. The paired CH3 domains of the Fc may comprise identical AB, CD and EF loops (of sequences identified above), and optionally both CH3 domains of the Fc are identical. The Fc, and optionally the whole multispecific antibody, may be homodimeric.

The multispecific antibody preferably comprises two heavy chains and two light chains. The two heavy chains comprise paired Fc regions, and a VH and CH1 region. A full heavy chain comprises VH linked sequentially to CH1, CH2 and CH3 respectively (in N to C direction) with a hinge region between domains CH1 and CH2. A full light chain comprises a VL linked to CL (in N to C direction). Each heavy chain is paired with a light chain, wherein VH associates with VL and CH1 associates with CL. The whole antibody thus comprises two Fabs, each Fab comprising a VH-VL domain pair.

The antibody may be a mAb² comprising two ICOS-binding Fab domains and an Fc region comprising two binding sites for PD-L1 (i.e., a PD-L1 binding Fcab), as illustrated in FIG. 3 .

Antibodies according to the present invention have shown interesting properties that indicate their suitability for use in therapy, offering the opportunity to treat human patients for diseases such as cancer. The antibodies may thus be provided with human heavy and light chain sequences, to minimise immunogenicity.

Thus, the Fc region is preferably a human Fc region. IgG Fc is commonly used in therapeutic molecules. The CH3 domain (and preferably the Fc as a whole) is preferably a human CH3 or human Fc that has been engineered to comprise the PD-L1 binding site. Thus, the PD-L1 binding site is provided by a CH3 domain comprising the AB, CD and EF loop sequences as indicated.

A range of antibody constant region sequences are known and the skilled person may select any to use for the multispecific antibody, engineering the structural loop region accordingly. A number of example constant regions are provided herein. It may be advantageous to use an Fc region that is effector positive, e.g., which has ADCC or CDC activity. Effector null sequences are also available. A preferred CH3 is a human IgG1 Fc CH3 domain (e.g., comprising the CH3 domain sequence of a human IgG1 sequence shown or mentioned herein). A preferred Fc is a human IgG1 Fc (e.g., comprising a human IgG1 sequence shown or mentioned herein). The multispecific antibody may comprise a CH3 domain having an amino acid sequence at least 90% identical (e.g., at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99 identical) to SEQ ID NO: 616. The multispecific antibody may comprise a CH3 domain having amino acid sequence SEQ ID NO: 616. The multispecific antibody may comprise an Fc region having an amino acid sequence at least 90% identical (e.g., at least 95 identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical) to SEQ ID NO: 620. The multispecific antibody may comprise a CH3 domain having amino acid sequence SEQ ID NO: 620.

The multispecific antibody may comprise two Fabs, which may be identical or different. One or both Fabs may bind ICOS. Where both Fabs comprise a binding site for ICOS, that binding site may be identical or different. The mAb2 molecule may thus be engineered to comprise two ICOS binding sites, wherein the two ICOS binding sites are identical or different. The two ICOS binding sites may bind the same or different epitopes of ICOS. Where the binding sites are identical, the two Fabs may comprise identical VH-VL domain pairs. Features and example sequences of anti-ICOS Fabs are described in more detail elsewhere herein. A preferred Fab comprises a VH-VL domain pair comprising the CDRs of antibody STIM003. It may comprise the STIM003 VH and/or STIM003 VL domain sequences or variants thereof as described herein. Other Fabs may be used, and a Fab of the multispecific antibody may comprise the VH and VL domain of any anti-ICOS antibody described herein. Optionally, the multispecific antibody comprises two anti-ICOS Fabs. In embodiments where non-identical Fabs are used, each Fab may be independently selected from Fabs comprising the VH and VL domains of the anti-ICOS antibodies described herein.

Both ICOS and PD-L1 are expressed following primary T cell activation. PD-L1 negatively regulates T cell activation, and inhibition of PD-L1 signalling has been clinically validated as an approach to upregulate the T cell immune response against tumour cells. In context, parallel depletion of ICOS-high Tregs and stimulation of ICOS-low effector T cells can enhance T cell activation to promote anti-tumour activity.

A multispecific antigen-binding molecule that blocks the negative regulatory activity of PD-L1 on PD1+ T cells and enhances T cell activation by delivering a positive signal through ICOS offers therapeutic potential in treating cancer and other conditions in which it is desirable to upregulate the T cell immune response. The fate of T cells in the tumour microenvironment and in tumour-draining lymph nodes is influenced by a balance of inhibitory and activatory receptors, and a molecule that binds and inhibits PD-L1 while acting as an ICOS agonist may effectively turn a negative signal (from the inhibitory PD-L1 receptor) into a positive signal (from the ICOS co-activatory receptor). The immune synapse between a T cell and an antigen-presenting cell (APC) or tumour cell can be envisaged as a receptor-dense space in which the balance of receptor occupancy determines signalling within the T cell, this receptor occupancy being governed by the identity and concentration of receptors being presented on the surface of the engaging APC/tumour cell. A multispecific molecule bearing a binding site for ICOS and a binding site for PD-L1 may act directly at this immune synapse to change the balance of signals received by T cells, shifting the balance towards activation of TEffs. Combination of anti-PD-L1 and anti-ICOS in one multispecific antigen-binding molecule, rather than separate antigen-binding molecules, provides a single agent that can act as a molecular switch. The multispecific molecule may cross-link ICOS and PD-L1 on different cells (FIG. 1 ).

Accordingly, further aspects of the invention relate to compositions comprising the multispecific antibody with one or more pharmaceutically acceptable excipients, optionally formulated for administration to a human patient by injection (e.g., intravenous or subcutaneous injection), for which example formulations and administration methods are disclosed. Further aspects relate to use of the antibodies in methods of treatment of the human or animal body by therapy, including treatment of cancer. Cancer treated in accordance with the present invention may be cancer that is associated with Tregs and/or that tests positive for expression of ICOS and FOXP3. The cancer may be head and neck cancer, non small cell lung cancer, cervical cancer, or another type of cancer disclosed herein.

In addition to binding its two cognate antigens, a multi-specific antigen-binding molecule may incorporate other moieties such as antibody effector regions to recruit cell-killing functions, which may further tip the immune balance towards T cell activation and killing of cancer cells, e.g., via depletion of TRegs which highly express ICOS on the cell surface and/or depletion of cancer cells expressing PD-L1. A bispecific antibody binding to ICOS and PD-L1 may trigger ADCC towards PD-L1+ immunosuppressive cells (e.g., MDSC, tumour cells) and/or ADCC towards ICOS+ immunosuppressive cells (e.g., Tregs).

A multispecific antigen-binding molecule that binds ICOS and PD-L1 may increase response rates of tumours that are already responsive to PD-L1 or ICOS monotherapy, increasing the proportion of patients in whom an anti-tumour response is observed and potentially improving the level of response, reducing tumour growth and extending survival compared with monotherapy. Some tumours are unresponsive to either anti-ICOS or anti-PD-L1 antibody, but may respond to a multispecific antibody that binds ICOS and PD-L1. Anti-ICOS/anti-PD-L1 bispecific binding molecules may also be used for inducing long term memory to antigens, e.g., tumour antigens, thereby providing protection against tumour regrowth. Thus, the multispecific approach described here offers advantages in improving response rates, duration of response, and patient survival, in the context of cancer therapy. Furthermore, a multispecific antigen-binding molecule can be administered to patients using simpler treatment regimens compared with multiple separate formulations of different therapeutic agents. A number of combination therapies are described herein, including administration of the multispecific antibody to a patient who is also receiving or has received treatment with a CTLA-4 antagonist or an anti-CTLA-4 antibody (e.g., ipilimumab or tremelimumab), or with a PD-1 antagonist or an anti-PD-1 antibody (e.g., pembrolizumab, nivolumab or genolimzumab).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Redirecting modulation of an immune checkpoint. The multispecific antigen-binding molecule effects a simultaneous blockade of PD-L1 receptors on antigen-presenting cells (APC) or tumour cells and agonism of the ICOS receptor on T effector cells, switching a negative regulatory signal to a positive regulatory signal at the T cell immune synapse.

FIG. 2 Alignment of amino acid sequences of CH1, CH2 and CH3 domains, numbered according to IMGT. Sequence comprising Fcab engineered to contain a binding site for PD-L1 is shown compared with wild type human IgG1 sequence. Fcab comprises PD-L1 binding site provided by loops AB (IMGT residues 14-18), CD (IMGT residues 45.1-78) and EF (IMGT residues 92-101) within the human IgG1 CH3 domain.

-   -   WT and Fcab CH1 sequence SEQ ID NO: 611     -   WT and Fcab CH2 sequence SEQ ID NO: 613     -   WT CH3 sequence SEQ ID NO: 615     -   Fcab CH3 sequence SEQ ID NO: 616

FIG. 3 . Example mAb² IgG format of bispecific antibody that binds ICOS and PD-L1. The mAb² is a homodimeric IgG comprising two anti-ICOS Fab and two CH3 domains each having three binding loops forming a PD-L1 binding site (the anti-PD-L1 Fcab region).

FIGS. 4A-4D (FIG. 4A) STIM001 and STIM003 mAb² binding to recombinant human ICOS protein. Data representative of three experiments. (FIG. 4B) STIM001 and STIM003 mAb² binding to recombinant mouse ICOS protein. Data representative of three experiments. (FIG. 4C) Human STIM001 and STIM003 mAb² binding to recombinant human PD-L1 protein. Data representative of three experiments. (FIG. 4D) Mouse STIM001 and STIM003 mAb² binding to recombinant mouse PD-L1 protein. Data representative of 3 experiments.

FIGS. 5A-5B Results of ICOS FACS binding assay described in Example 4. FIG. 5A) mAb² binding to human ICOS expressed on CHO cells. Data representative of 3 experiments. FIG. 5B) mAb² binding to mouse ICOS expressed on CHO cells. Data representative of 3 experiments.

FIG. 6 Results of human PD-L1 FACS binding assay described in Example 4. A) Human PD-L1-binding FACS with anti-human IgG detection. Binding profiles of STIM001_289, STIM003_289 and IgG1_289 anti-PD-L1 mAb² s and respective mAb controls. B) Human PD-L1-binding FACS with bound human ICOS labelled AlexaFluor 647 detection. Binding profiles of STIM001_289, STIM003_289 and IgG1_289 anti-PD-L1 mAb² s and respective mAb controls.

FIG. 7 Results of mouse PD-L1 FACS binding assay described in Example 4. A) Mouse PD-L1-binding FACS with anti-human IgG detection. Binding profiles of STIM001_457, STIM003_457 and IgG1_438 anti-PD-L1 mAb² s and respective monospecific mAb controls. B) Mouse PD-L1-binding FACS with bound human ICOS labelled AlexaFluor 647 detection. Binding profiles of STIM001_457, STIM003_457 and IgG1_438 anti-PD-L1 mAb² s and respective monospecific mAb controls.

FIGS. 8A-8B (FIG. 8A) Human STIM001 and STIM003 mAb² Fc engagement to human FcγRIIIa on effector cells, as described in Example 5b. Data representative of 3 experiments. (FIG. 8B) Mouse STIM001 and STIM003 mAb² Fc engagement to FcγRIIIa on effector cells, as described in Example 5b. Data representative of 3 experiments.

FIGS. 9A-9C Concentration-dependent study of STIM001_289 and STIM003_289 mediated ADCC on ICOS-transfected CCRF-CEM cells using freshly isolated NK cells as effector cells for 3 independent donors, as described in Example 5c. (FIG. 9A) Results of study on cells from donor 334. (FIG. 9B) Results of study on cells from donor 326. (FIG. 9C) Results of study on cells from donor 341. The effector cells and target cells (effector:target ratio of 5:1) were incubated together with antibody for 4 hours. Dye release from lysed target cells was measured as described in the kit manufacturer's instructions. Lysis buffer was used to determine the 100% release. Basal killing (no Ab) is indicated by a dotted line at the bottom of each graph.

FIG. 10 A) Results of mouse ICOS-Ligand neutralisation HTRF assay with mouse ICOS receptor described in Example 6. Neutralisation profiles of STIM001_289, STIM001_457, STIM003_289 and STIM003_457 mAb². Data representative of three experiments. B) Results of human ICOS-Ligand neutralisation HTRF assay with human ICOS receptor described in Example 6. Neutralisation profiles of STIM001_289, STIM001_457, STIM003_289 and STIM003_457 mAb². Data representative of three experiments.

FIG. 11 Results of PD-L1 neutralisation assay described in Example 7. A) Human PD-L1 Neutralisation FACS to human PD1. Binding profiles of STIM001_289, STIM003_289 and IgG1_289 anti-PD-L1 mAb² s and respective mAb controls. B) Human PD-L1 Neutralisation FACS to human CD80. Binding profiles of STIM001_289, STIM003_289 and IgG1_289 anti-PD-L1 mAb² s and respective mAb controls.

FIG. 12 A) Data from mouse PD-L1 neutralisation assay (FACS) to mouse PD1 as described in Example 7. Binding profiles of STIM001_457, STIM003_457 and IgG1_438 anti-PD-L1 mAb² s and respective controls. B) Data from mouse PD-L1 neutralisation assay (FACS) to mouse CD80. Binding profiles of STIM001_457, STIM003_457 and IgG1_438 anti-PD-L1 mAb² s and respective controls.

FIGS. 13A-13C Concentration-dependent study of STIM001_289 and STIM003_289 vs STIM001 and STIM003 agonist effect on isolated human T-cells co-stimulated with CD3/CD28 dynabeads for 3-days. IFN-γ production was used as a read-out of ICOS agonism. All antibodies were tested plate-bound and compared to their isotype controls. (FIG. 13A) Mean±SD values of technical replicate 1 as well as non-linear regression curves (variable slope, 4-parameter) are shown for donor 278. (FIG. 13B) Mean±SD values of technical replicate 2 as well as non-linear regression curves (variable slope, 4-parameter) are shown for 1 donor (278). (FIG. 13C) An example set of data comparing the levels of IFN-γ (mean value) induced at one given dose (3.3 μM) for all 4 donors. Each dot represents an independent donor identifiable by its number and the median of 4 donors is marked by a line. Significance was assessed using Friedman statistic test and p-values are indicated on the graph.

FIGS. 14A-14B Concentration-dependent study of STIM001_289, STIM003_289 and IgG1_289 vs PD-L1 AbV effect on cytokine production by CD45RO⁺ T-cells co-culture with autologous monocytes in presence of CD3 antibody (TCR activation). In this assay, as described in Example 9, IFN-γ production is used as a read-out of the neutralisation of PD-1/PD-L1 interaction by the test antibody. All antibodies were compared to the isotype control (IgG1). Raw data of one independent donor (288) is shown in the upper panel. The basal IFN-γ levels (mono/T-cells dotted line) was used to normalise the values and calculate the fold increase in IFN-γ. In the lower panel is shown an example of data comparing the increase in IFN-γ induced at one given dose (10 nM) for all 7 donors. Each dot represents an independent donor identifiable by its number and the median is marked by a line. Significance was assessed using Friedman statistic test (*<0.05 and **<0.01).

FIG. 15 Effect of STIM001_457 and STIM003_457 bispecific antibodies in the J558 syngeneic tumour study described in Example 10. Each treatment group is represented by a “spider plot” showing the tumour size of individual animals (n=10 or n=8 per group). Both bispecific antibodies demonstrated significant anti-tumour efficacy with 5 out 8 animals treated with STIM001_457 (B) and 4 out of 8 of those treated with STIM003_457 (C) cured from their disease at day 37 compared to the saline treated control group (A). The number of animals cured of their disease is indicated on the bottom right of the respective graphs. Scheduled dosing days are indicated by dotted lines (day 11, 15, 18, 22, 25 and 29).

FIG. 16 Kaplan-Meier survival curves/time on study of the % mice surviving after the different treatments described in Example 10. Median survival time of animals on saline (open square) and STIM003_457 (triangle) were 18 and 27.5 days, respectively. Median survival for STIM001_457 (black circle) was not reached.

FIGS. 17A-17D Data from CT26 in vivo efficacy study described in Example 11a. Each treatment group is represented by a “spider plot” showing the tumour size of individual animals (n=10 per groups). For each group, the number of animals cured of their disease is indicated on the bottom left of the respective graphs. Dosing was on days 6, 8, 10, 13, 15 and 17, and dosing time is indicated by the shaded area. (FIG. 17A) Tumor size of animals treated with saline; (FIG. 17B) Tumor size of animals treated with IgG1_457 LAGA control. (FIG. 17C) Tumor size of animals treated with STIM003_457. (FIG. 17D) Tumor size of animals treated with STIM001_457.

FIG. 18 Kaplan Meier plot for CT26 study described in Example 11a. Circles: saline control. Squares: IgG1_487 control. Up triangles: STIM003_457. Down triangles: STIM001_457.

FIG. 19 Treatment with ICOS/PD-L1 antibody results in a long-term anti-tumour memory response in animals previously cured from CT26 tumours. As described in Example 11b, mice cured from CT26 colon cancer were rechallenged s.c. in the left side of their abdomen with either 2.5×10⁵ EMT-6 cells (n=4 mice per group) or 1×10⁵ CT26 cells (n=5 mice per group). The spider plots show the tumour growth during 20 days following EMT-6 or CT26 cell inoculation.

FIGS. 20A-20D Results of A20 in vivo efficacy study described in Example 12. Each treatment group is represented by a “spider plot” showing the tumour size of individual animals (n=10 per group). For each group, the number of animals cured of their disease is indicated on the bottom left of the respective graph. Dosing was on days 8, 11, 15, 18, 22 and 25. (FIG. 20A) Tumor size of animals treated with saline. (FIG. 20B) Tumor size of animals treated with IgG1_457. (FIG. 20C) Tumor size of animals treated with STIM003_457. (FIG. 20D) Tumor size of animals treated with STIM001_457.

FIG. 21 Results of A20 in vivo efficacy study described in Example 12. The humane endpoint survival statistics were calculated from the Kaplan-Meier curves using GraphPad Prism V7.0. This approach was used to determine if specific treatments were associated with improved survival.

FIGS. 22A-22D Data from EMT6 in vivo efficacy study described in Example 13. Each treatment group is represented by a “spider plot” showing the tumour size of individual animals (n=10 per group). FIG. 22A) Tumor size of animals treated with saline. FIG. 22B) Tumor size of animals treated with anti-PD-L1 mAb² control antibody. FIG. 22C) Tumor size of animals treated with STIM003_457. FIG. 22D) Tumor size of animals treated with STIM001_457. For each group, the number of animals cured of their disease is indicated on the bottom left of the respective graph. Dosing was on days 6, 9, 13, 16, 20 and 23.

FIG. 23 Survival (time on study) for the animals treated with saline (black circles), anti-PD-L1 mAb² control antibody (squares), STIM003_457 (up triangles), or STIM001_457 (down triangles), as described in Example 13. Both ICOS/PD-L1 bispecific antibodies significantly improved the overall survival of animals compared with those treated with saline.

FIGS. 24A-24D Bispecific efficacy in the EMT6 model described in Example 13. FIG. 24A) Tumor volume when treated with IgG1 LAGA hybrid control mAb² antibody with anti-PD-L1 457 Fcab. FIG. 24B) Tumor volume when treated with a combination of STIM003 and anti-PD-L1 antibody (mouse IgG2a format). FIG. 24C) Tumor volume when treated with STIM001_457 bispecific antibody. FIG. 24D) Tumor volume when treated with STIM003_457 bispecific antibody.

FIG. 25 Kaplan Meier (humane endpoint) showing superior efficacy of treatment with the PD-L1/ICOS bispecific antibodies (down triangles for STIM001_457, up triangles for STIM003_457) compared with combined administration of anti-PD-L1 monospecific antibody and anti-ICOS monospecific antibody (black diamonds) in the EMT6 model described in Example 13. Data from saline control treatments shown in closed circles. Data from IgG1 LAGA hybrid control mAb² antibody with anti-PD-L1 457 Fcab shown in open squares.

FIGS. 26A-26C Representative example from two independent experiments in the PD-L1 dependent ICOS agonism assay reported in Example 14. (FIG. 26A) Percent effect of culturing MJ cells in wells coated with BSA. (FIG. 26B) Percent effect of culturing MJ cells in wells coated with B7-H1-Fc. (FIG. 26C) Percent effect of culturing MJ cells in wells coated with Goat anti-human IgG Fcg fragment specific F(ab′)2.

FIG. 27 Identification of four different quadrants on dot plot graph for mAb² antibodies in a PD-L1/ICOS cell recruitment assay by flow cytometry.

FIG. 28 Titration of mAb² and monospecific antibodies in a PD-L1/ICOS cell recruitment assay by flow cytometry. CHO human PD-L1 and CHO human ICOS were stained with CellTrace™ Far Red and CellTrace™ Violet respectively and incubated together in presence of antibodies for an hour prior to the detection of fluorescence and identification of double positive population. Data shown are representative of two independent experiments.

FIGS. 29A-29D FACS analysis revealed that STIM003_457 and STIM001_457 significantly deplete regulatory T-cells (T_(Regs)) and increase effector cell: T_(Regs) ratio in tumour. Animals (BALB/c mice) were dosed with saline, STIM003_457 or STIM001_457 (n=8 per group) on days 13 and 15 days post-implantation of CT-26.WT tumour cells s/c. Proportion of T_(Regs) of total live tumour cells (FIG. 29A) and of total CD4⁺ cells (FIG. 29B) were significantly decreased in response to both antibodies compared to saline. Ratios of CD4⁺ effector cells to T_(Regs) (FIG. 29C), and of CD8⁺ cells to T_(Regs) (FIG. 29D) are significantly increased compared to the control. Kruskal-Wallis test was performed followed by post-hoc Dunn's test. * p<0.05. ** p<0.01. *** p<0.001. **** p<0.0001.

FIGS. 30A-30D FACS analysis shows that STIM003_457 and STIM001_457 have little effect on regulatory T-cell (T_(Regs)) levels in the spleen of a CT-26.WT tumour-bearing mouse. STIM003_457 shows a marginal T_(Regs) depletion as a percentage of total live cells, but STIM001_457 has no effect (FIG. 30A). No clear changes were observed when looking at the effect of the bispecific on T_(Regs) as a percentage of total CD4⁺ cells (FIG. 30B). No significant changes are seen in CD4⁺ effector cell to T_(Regs) (FIG. 30C), and CD8⁺ cell to T_(Regs) ratios (FIG. 30D). Kruskal-Wallis test was performed followed by post-hoc Dunn's test. * p<0.05.

FIG. 31 FACS analysis demonstrates increase in ICOS-Ligand (ICOS-L) expression on B-cells in the spleens of CT26-WT tumour-bearing mice dosed with STIM003_457 and STIM001_457. When compared to saline, both bispecific antibodies caused a significant increase in the percentage of B-cells expressing ICOS-L in the spleen (A). A significant increase in mean fluorescence intensity (relative expression) of ICOS-L on B-cells was also seen in both bi-specific groups compared to the saline group (B). Kruskal-Wallis test was performed followed by post-hoc Dunn's test. * p<0.05. **** p<0.0001.

FIG. 32 Graph showing the average weight of the mice over the 46 days for the different treatment groups. Vertical lines indicate the day the animals were dosed IP. Note that the small decrease in average weight observed from day for group 3 (aCTLA-4 monotherapy) and group 7 (STIM003/aPDL1 combination) is due to some animals coming out of the study for tumour size.

FIGS. 33A-33G Spider plot graphs showing the CT26 tumour size of individual animals over time in response to the different treatments. The “triple combination” of antibodies (against ICOS, PD-L1 and PD1 or CTLA-4) were associated with the most pronounced anti-tumour response in the CT26 model. Vertical lines indicate the day the animals were dosed IP. For the combination the antibodies were injected concomitantly. The numbers at the bottom right end of each graph indicate the number of animals still on study on day 46 (40 days after the treatments were initiated).

FIGS. 34A-34D Progression of tumour volume over time in mice treated with (i) saline, (ii) anti-CTLA-4 antibody 9D9, (iii) anti-ICOS/anti-PD-L1 bispecific antibody, (iv) combination of anti-CTLA-4 antibody 9D9 and anti-ICOS/anti-PD-L1 bispecific antibody. Dotted vertical lines indicate dosing days.

FIG. 35 Kaplan Meier plot showing superior efficacy of PD-L1/ICOS bispecific antibody combined with anti-CTLA-4 9D9 monospecific antibody, compared with either the bispecific or monospecific antibody administered alone in a CT26 tumour study.

DETAILED DESCRIPTION Antibodies

Digestion of antibodies with the enzyme papain results in two identical antigen-binding fragments, known also as “Fab” fragments, and a “Fc” fragment, having no antigen-binding activity but having the ability to crystallize. “Fab” when used herein refers to a fragment of an antibody that includes one constant and one variable domain of each of the heavy and light chains. The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The “Fc fragment” refers to the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognised by Fc receptors (FcR) found on certain types of cells. Digestion of antibodies with the enzyme pepsin, results in the F(ab′)2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab′)2 fragment has the ability to crosslink antigen.

A mAb² comprises a V_(H) and V_(L) domain from an intact antibody, fused to a modified constant region, which has been engineered to form an antigen-binding site, known as an “Fcab”. The technology behind the Fcab/mAb² format is described in more detail in WO2008/003103, and the description of the mAb² format is incorporated herein by reference. Further descriptions of this format can be found in WO2006/072620, WO2008/003116, WO2009/000006 and WO2009/0132876.

“Fv” when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent or covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognise and bind antigen, although at a lower affinity than the entire binding site.

In a Fab, an antibody antigen-binding site may be provided by one or more antibody variable domains. In an example, the antibody binding site is provided by a single variable domain, e.g., a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, the binding site comprises a VH/VL pair or two or more of such pairs. Thus, an antibody antigen-binding site may comprise a VH and a VL.

An antibody normally comprises an antibody VH and/or VL domain. Isolated VH and VL domains of antibodies are also part of the invention. The antibody variable domains are the portions of the light and heavy chains of antibodies that include amino acid sequences of complementarity determining regions (CDRs; ie., CDR1, CDR2, and CDR3), and framework regions (FRs). Thus, within each of the VH and VL domains are CDRs and FRs. A VH domain comprises a set of HCDRs, and a VL domain comprises a set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to the methods used in this invention, the amino acid positions assigned to CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to IMGT nomenclature. An antibody may comprise an antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It may alternatively or also comprise an antibody VL domain comprising a VL CDR1, CDR2 and CDR3 and a framework. Examples of antibody VH and VL domains and CDRs according to the present invention are as listed in the appended sequence listing and tables that form part of the present disclosure. The IMGT system is as described by LeFranc²³. All VH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs and sets of LCDRs disclosed herein represent aspects and embodiments of the invention. As described herein, a “set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a “set of CDRs” includes HCDRs and LCDRs.

The term “hypervariable region”, “CDR region” or “CDR” refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antigen binding sites of an antibody include six hypervariable regions: three in the VH (CDRH1, CDRH2, CDRH3), and three in the VL (CDRL1, CDRL2, CDRL3). These regions of the heavy and light chains of an antibody confer antigen-binding specificity to the antibody. CDRs may be defined according to the Kabat system (see Kabat, E. A. et al., 1991, “Sequences of Proteins of Immunological Interest”, 5th edit., NIH Publication no. 91-3242, U.S. Department of Health and Human Services). Other systems may be used to define CDRs, which as the system devised by Chothia et al (see Chothia, C. & Lesk, A. M., 1987, “Canonical structures for the hypervariable regions of immunoglobulins”, J. Mol. Biol., 196, 901-917) and the IMGT system (see Lefranc, M. P., 1997, “Unique database numbering system for immunogenetic analysis”, Immunol. Today, 18, 50). An antibody typically contains 3 heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRs is used here to indicate one or several of these regions. A person skilled in the art is able to readily compare the different systems of nomenclature and determine whether a particular sequence may be defined as a CDR.

A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies and specifically excludes a humanized antibody comprising non-human antigen-binding residues. The term “specifically binds to” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA).

The term “effector function” as used herein is meant to refer to one or more of antibody dependent cell mediated cytotoxic activity (ADCC), complement-dependent cytotoxic activity (CDC) mediated responses, Fc-mediated phagocytosis or antibody dependent cellular phagocytosis (ADCP) and antibody recycling via the FcRn receptor.

The term “heavy chain” when used with reference to an antibody refers to five distinct types, called alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the heavy chain constant domain. These distinct types of heavy chains are well known and give rise to five classes of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG1, IgG3 and IgG4. Preferably the heavy chain is a human heavy chain. In the human population, multiple heavy chain constant region alleles, of each immunoglobulin or immunoglobulin subclass, exist. The nucleotide and amino acid sequences of these allelic variants are accessible on publicly available databases such as IMGT, ENSEMBL Swiss-Prot and Uniprot. Allelic variants may also be identified in various genome sequencing projects. In one embodiment, the antibodies and antibody fragments disclosed herein comprise a heavy chain encoded by a IgG1 constant region allele, which includes, but is not limited to, human IGHG1*01 (Seq ID Nos: 340, 341 & 537), IGHG1*02 (Seq ID Nos: 340, & 341 & 537), IGHG1*03 (Seq ID Nos: 523 & 524), IGHG1*04 (Seq ID Nos: 525 & 526) and IGHG1*05 (Seq ID Nos: 340, 341 & 537). In one embodiment, the antibodies and antibody fragments disclosed herein comprise a protein encoded by a IgG2 constant region allele, which includes, but is not limited to, human IGHG2*01 (Seq ID Nos: 527 & 528), IGHG2*02 (Seq ID Nos: 529 & 530), IGHG2*03 (Seq ID Nos: 527 & 528), IGHG2*04 (Seq ID Nos: 531 & 532), IGHG2*05 (Seq ID Nos: 527 & 528) and IGHG2*06 (Seq ID Nos: 533 & 534). In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by an IgG3 constant region allele, which includes but is not limited to human IGHG3*01, IGHG3*02, IGHG3*03, IGHG3*04, IGHG3*05, IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*09, IGHG3*10, IGHG3*11, IGHG3*12, IGHG3*13, IGHG3*14, IGHG3*15, IGHG3*16, IGHG3*17, IGHG3*18 and IGHG3*19. In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by a IgG4 constant region allele, which includes but is not limited to human IGHG4*01 (Seq ID Nos: 192 & 193), IGHG4*02 (Seq ID Nos: 194 & 195), IGHG4*03 (Seq ID Nos: 196 & 197) and IGHG4*04 (Seq ID Nos: 192 & 193). In another example, the heavy chain is a disabled IgG isotype, e.g. a disabled IgG4. In certain embodiments, the antibodies of the invention comprise a human gamma 4 constant region. In another embodiment, the heavy chain constant region does not bind Fc-γ receptors, and e.g. comprises a Leu235Glu mutation. In another embodiment, the heavy chain constant region comprises a Ser228Pro mutation to increase stability. In another embodiment, the heavy chain constant region is IgG4-PE (SEQ ID NO: 199). In another embodiment, the antibodies and antibody fragments disclosed herein comprise a heavy chain constant region encoded by a murine IgG1 constant region allele, which includes but is not limited to mouse IGHG1*01 or IGHG1*02. In one embodiment, the antibodies and antibody fragments disclosed herein comprise a heavy chain constant region encoded by a murine IgG2 constant region allele, which includes, but is not limited to, mouse IGHG2A*01, IGHG2A*02, IGHG2B*01, IGHG2B*02, IGHG2C*01, IGHG2C*02 or IGHG2C*03. In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by a murine IgG3 constant region allele, which includes but is not limited to mouse IGHG3*01.

An “isolated” or “purified” antibody or protein is one that has been identified, separated and/or recovered from a component of its production environment (e.g., natural or recombinant). For example, the antibody or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”). When the antibody is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the antibody have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a preferred embodiment, antibodies of the invention are isolated or purified.

The terms “Kabat numbering,” and like terms are recognized in the art and refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region typically ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3.

The term “light chain” when used in reference to an antibody refers to the immunoglobulin light chains, of which there are two types in mammals, lambda (λ) and kappa (κ). Preferably, the light chain is a human light chain. Preferably the light chain constant region is a human constant region. In the human population, multiple light chain constant region alleles exist. The nucleotide and amino acid sequences of these allelic variants are accessible on publicly available databases such as IMGT, ENSEMBL, Swiss-Prot and Uniprot. In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by a human K constant region allele, which includes, but is not limited to, IGKC*01 (Seq ID Nos:206 & 207), IGKC*02 (Seq ID Nos:208 & 209), IGKC*03 (Seq ID Nos:210 & 211), IGKC*04 (Seq ID Nos:212 & 213) and IGKC*05 (Seq ID Nos:214 & 215). In one embodiment, the antibodies or antibody fragments disclosed herein comprise a protein encoded by a human A constant region allele, which includes but is not limited to IGLC1*01 (Seq ID Nos:216 & 217), IGLC1*02 (Seq ID Nos:218, 219 & 220), IGLC2*01 (Seq ID Nos:221, 222 & 538), IGLC2*02 (Seq ID Nos:224 & 225), IGLC2*03 (Seq ID Nos:224 & 225), IGLC3*01 (Seq ID Nos:226 & 227), IGLC3*02 (Seq ID Nos:228 & 229), IGLC3*03 (Seq ID Nos:230 & 231), IGLC3*04 (Seq ID Nos:232 & 233), IGLC6*01 (Seq ID Nos:234 & 235), IGLC7*01 (Seq ID Nos:236 & 237), IGLC7*02 (Seq ID Nos:236 & 237), IGLC7*03 (Seq ID Nos:535 & 536). In another embodiment, the antibodies and antibody fragments disclosed herein comprise a light chain constant region encoded by a mouse K constant region allele, which includes, but is not limited to, IGKC*01, IGKC*03 or IGKC*03. In another embodiment, the antibodies and antibody fragments disclosed herein comprise a light chain constant region encoded by a mouse A constant region allele, which includes, but is not limited to, IGLC1*01, IGLC2*01 or IGLC3*01.

The term “variable region” or “variable domain” refers to a portion of the light and heavy chains, typically about the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complimentarily determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). The CDRs of the heavy chains are primarily responsible for the interaction of the antibody with antigen. In preferred embodiments, the variable region is a human variable region.

An antibody may comprise a VH domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VH domain of any of the antibodies shown in the appended sequence listing, and/or comprising a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain of any of those antibodies. Algorithms that can be used to calculate % identity of two amino acid sequences include e.g. BLAST, FASTA, or the Smith-Waterman algorithm, e.g. employing default parameters. Particular variants may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue).

Alterations may be made in one or more framework regions and/or one or more CDRs. Variants are optionally provided by CDR mutagenesis. The alterations normally do not result in loss of function, so an antibody comprising a thus-altered amino acid sequence may retain an ability to bind ICOS. It may retain the same quantitative binding ability as an antibody in which the alteration is not made, e.g. as measured in an assay described herein. The antibody comprising a thus-altered amino acid sequence may have an improved ability to bind ICOS.

Alteration may comprise replacing one or more amino acid residue with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residue into a non-naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acid into the sequence. Examples of numbers and locations of alterations in sequences of the invention are described elsewhere herein. Naturally occurring amino acids include the 20 “standard” L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter codes. Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from modification of an existing amino acid residue. Non-standard amino acids may be naturally occurring or non-naturally occurring.

The term “variant” as used herein refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, substitutions or additions, yet retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as “conservative”, in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention may also be “non-conservative”, in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid. In some embodiments amino acid substitutions are conservative. Also encompassed within the term variant when used with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide).

In some aspects, one can use “synthetic variants”, “recombinant variants”, or “chemically modified” polynucleotide variants or polypeptide variants isolated or generated using methods well known in the art. “Modified variants” can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Some aspects use include insertion variants, deletion variants or substituted variants with substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins. The term “conservative substitution,” when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.). Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered “conservative substitutions” if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents.

One can select the amino acid that will substitute an existing amino acid based on the location of the existing amino acid, including its exposure to solvents (i.e., if the amino acid is exposed to solvents or is present on the outer surface of the peptide or polypeptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol. 119(1986); 205-218 and S. French and B. Robson, J. Mol. Evol. 19(1983)171. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent), for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.

In alternative embodiments, one can also select conservative amino acid substitutions encompassed suitable for amino acids on the interior of a protein or peptide, for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent), for example but not limited to, one can use the following conservative substitutions: where Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term of variants.

Antibodies disclosed herein may be modified to increase or decrease serum half-life. In one embodiment, one or more of the following mutations: T252L, T254S or T256F are introduced to increase biological half-life of the antibody. Biological half-life can also be increased by altering the heavy chain constant region CH₁ domain or CL region to contain a salvage receptor binding epitope taken from two loops of a CH₂ domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022, the modifications described therein are incorporated herein by reference. In another embodiment, the Fc hinge region of an antibody or antigen-binding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment. One or more amino acid mutations are introduced into the CH₂—CH₃ domain interface region of the Fc-hinge fragment such that the antibody or fragment has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. Other methods of increasing serum half-life are known to those skilled in the art. Thus, in one embodiment, the antibody or fragment is PEGylated. In another embodiment, the antibody or fragment is fused to an albumin-biding domain, e.g. an albumin binding single domain antibody (dAb). In another embodiment, the antibody or fragment is PASylated (i.e. genetic fusion of polypeptide sequences composed of PAS (XL-Protein GmbH) which forms uncharged random coil structures with large hydrodynamic volume). In another embodiment, the antibody or fragment is XTENylated®/rPEGylated (i.e. genetic fusion of non-exact repeat peptide sequence (Amunix, Versartis) to the therapeutic peptide). In another embodiment, the antibody or fragment is ELPylated (i.e. genetic fusion to ELP repeat sequence (PhaseBio)). These various half-life extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, which fusions, e.g. in Tables 2 and 6, are incorporated herein by reference.

The antibody may have a modified constant region which increases stability. Thus, in one embodiment, the heavy chain constant region comprises a Ser228Pro mutation. In another embodiment, the antibodies and fragments disclosed herein comprise a heavy chain hinge region that has been modified to alter the number of cysteine residues. This modification can be used to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

Fc Effector Functions, ADCC, ADCP and CDC

As discussed above, antibodies can be provided in various isotypes and with different constant regions. The skilled person will be able to select a suitable Fc region in which to provide the described binding site for PD-L1. Examples of human IgG antibody heavy chain constant region sequences are shown herein. The Fc region of the antibody primarily determines its effector function in terms of Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement dependent cytotoxicity (CDC) activity and antibody-dependent cell phagocytosis (ADCP) activity. These “cellular effector functions”, as distinct from effector T cell function, involve recruitment of cells bearing Fc receptors to the site of the target cells, resulting in killing of the antibody-bound cell. In addition to ADCC and CDC, the ADCP mechanism [24] represents a means of depleting antibody-bound T cells, and thus targeting high ICOS expressing TRegs for deletion.

Cellular effector functions ADCC, ADCP and/or CDC may also be exhibited by antibodies lacking Fc regions. Antibodies may comprise multiple different antigen-binding sites, one directed to ICOS and another directed to a target molecule where engagement of that target molecule induces ADCC, ADCP and/or CDC, e.g., an antibody comprising two scFv regions joined by a linker, where one scFv can engage an effector cell.

An antibody according to the present invention may be one that exhibits ADCC, ADCP and/or CDC. Alternatively, an antibody according to the present invention may lack ADCC, ADCP and/or CDC activity. In either case, an antibody according to the present invention may comprise, or may optionally lack, an Fc region that binds to one or more types of Fc receptor. Use of different antibody formats, and the presence or absence of FcR binding and cellular effector functions, allow the antibody to be tailored for use in particular therapeutic purposes as discussed elsewhere herein.

A suitable antibody format for some therapeutic applications employs a wild-type human IgG1 constant region. A constant region may be an effector-enabled IgG1 constant region, optionally having ADCC and/or CDC and/or ADCP activity. A suitable wild type human IgG1 constant region sequence is SEQ ID NO: 340 (IGHG1*01). Further examples of human IgG1 constant regions are shown herein. FIG. 2 shows the CH1, CH2 and CH3 regions of the Fc region present in the _289 mAb2 molecules used in the Examples, aligned against the corresponding domains of a human wild type IgG1 constant region. Variations of this may be made in the regions outside the binding loops AB, CD and EF, for example to alter the effector functions. Mutations (e.g., substitutions) may be made to match other immunoglobulin isotypes, or for other reasons. An example mutation is A38V, namely the substitution of valine for the alanine at IMGT residue 38.

For testing of candidate therapeutic antibodies in mouse models of human disease, an effector positive mouse constant region, such as mouse IgG2a (mIgG2a), may be included instead of an effector positive human constant region.

A constant region may be engineered for enhanced ADCC and/or CDC and/or ADCP.

The potency of Fc-mediated effects may be enhanced by engineering the Fc domain by various established techniques. Such methods increase the affinity for certain Fc-receptors, thus creating potential diverse profiles of activation enhancement. This can achieved by modification of one or several amino acid residues [25]. Human IgG1 constant regions containing specific mutations or altered glycosylation on residue Asn297 (e.g., N297Q, EU index numbering) have been shown to enhance binding to Fc receptors. Example mutations are one or more of the residues selected from 239, 332 and 330 for human IgG1 constant regions (or the equivalent positions in other IgG isotypes). An antibody may thus comprise a human IgG1 constant region having one or more mutations independently selected from N297Q, S239D, I332E and A330L (EU index numbering). A triple mutation (M252Y/S254T/T256E) may be used to enhance binding to FcRn, and other mutations affecting FcRn binding are discussed in Table 2 of [²⁶], any of which may be employed in the present invention.

Increased affinity for Fc receptors can also be achieved by altering the natural glycosylation profile of the Fc domain by, for example, generating under fucosylated or defucosylated variants [27]. Non-fucosylated antibodies harbour a tri-mannosyl core structure of complex-type N-glycans of Fc without fucose residue. These glycoengineered antibodies that lack core fucose residue from the Fc N-glycans may exhibit stronger ADCC than fucosylated equivalents due to enhancement of FcγRIIIa binding capacity. For example, to increase ADCC, residues in the hinge region can be altered to increase binding to Fc-gamma RIII [28]. Thus, an antibody may comprise a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to human Fcγ receptors selected from the group consisting of FcyRIIB and FcyRIIA with higher affinity than the wild type human IgG heavy chain constant region binds to the human Fcγ receptors. The antibody may comprise a human IgG heavy chain constant region that is a variant of a wild type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to human FcγRIIB with higher affinity than the wild type human IgG heavy chain constant region binds to human FcγRIIB. The variant human IgG heavy chain constant region can be a variant human IgG1, a variant human IgG2, or a variant human IgG4 heavy chain constant region. In one embodiment, the variant human IgG heavy chain constant region comprises one or more amino acid mutations selected from G236D, P238D, S239D, S267E, L328F, and L328E (EU index numbering system). In another embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F (EU index numbering system). The enhancement of CDC may be achieved by amino acid changes that increase affinity for C1q, the first component of the classic complement activation cascade [29]. Another approach is to create a chimeric Fc domain created from human IgG1 and human IgG3 segments that exploit the higher affinity of IgG3 for C1q [30]. Antibodies of the present invention may comprise mutated amino acids at residues 329, 331 and/or 322 to alter the C1q binding and/or reduced or abolished CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein may contain Fc regions with modifications at residues 231 and 239, whereby the amino acids are replaced to alter the ability of the antibody to fix complement. In one embodiment, the antibody or fragment has a constant region comprising one or more mutations selected from E345K, E430G, R344D and D356R, in particular a double mutation comprising R344D and D356R (EU index numbering system).

WO2008/137915 described anti-ICOS antibodies with modified Fc regions having enhanced effector function. The antibodies were reported to mediate enhanced ADCC activity as compared to the level of ADCC activity mediated by a parent antibody comprising the VH and VK domains and a wild type Fc region. Antibodies according to the present invention may employ such variant Fc regions having effector function as described therein.

ADCC activity of an antibody may be determined in an assay as described herein. ADCC activity of an anti-ICOS antibody may be determined in vitro using an ICOS positive T cell line as described herein. ADCC activity of an anti-PD-L1 antibody may be determined in vitro in an ADCC assay using PD-L1 expressing cells.

For certain applications (such as in the context of vaccination) it may be preferred to use antibodies without Fc effector function. Antibodies may be provided without a constant region, or without an Fc region—examples of such antibody formats are described elsewhere herein. Alternatively, an antibody may have a constant region which is effector null. An antibody may have a heavy chain constant region that does not bind Fcγ receptors, for example the constant region may comprise a Leu235Glu mutation (i.e., where the wild type leucine residue is mutated to a glutamic acid residue). Another optional mutation for a heavy chain constant region is Ser228Pro, which increases stability. A heavy chain constant region may be an IgG4 comprising both the Leu235Glu mutation and the Ser228Pro mutation. This “IgG4-PE” heavy chain constant region is effector null.

An alternative effector null human constant region is a disabled IgG1. A disabled IgG1 heavy chain constant region may contain alanine at position 235 and/or 237 (EU index numbering), e.g., it may be a IgG1*01 sequence comprising the L235A and/or G237A mutations (“LAGA”).

A variant human IgG heavy chain constant region may comprise one or more amino acid mutations that reduce the affinity of the IgG for human FcγRIIIA, human FcγRIIA, or human FcγRI. In one embodiment, the FcγRIIB is expressed on a cell selected from the group consisting of macrophages, monocytes, B-cells, dendritic cells, endothelial cells, and activated T-cells. In one embodiment, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T; S239D and I332E; S239D, A330L, and I332E; S298A, E333A, and K334A; G236A, S239D, and I332E; and F243L, R292P, Y300L, V305I, and P396L (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises a S239D, A330L, or I332E amino acid mutations (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises an S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region is a variant human IgG1 heavy chain constant region comprising the S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the antibody or fragment comprises an afucosylated Fc region. In another embodiment, the antibody or fragment thereof is defucosylated. In another embodiment, the antibody or fragment is under fucosylated.

An antibody may have a heavy chain constant region that binds one or more types of Fc receptor but does not induce cellular effector functions, i.e., does not mediate ADCC, CDC or ADCP activity. Such a constant region may be unable to bind the particular Fc receptor(s) responsible for triggering ADCC, CDC or ADCP activity.

ICOS

“ICOS” or “the ICOS receptor” referred to herein may be human ICOS, unless the context dictates otherwise. Sequences of human, cynomolgus and mouse ICOS are shown in the appended sequence listing, and are available from NCBI as human NCBI ID: NP_036224.1, mouse NCBI ID: NP_059508.2 and cynomolgus GenBank ID: EHH55098.1.

The ICOS ligand (ICOSL, also known as B7-H2) is a cell surface expressed molecule that binds to the ICOS receptor [31]. This intercellular ligand-receptor interaction promotes multimerisation of ICOS on the T cell surface, activating the receptor and stimulating downstream signalling in the T cell. In effector T cells, this receptor activation stimulates the effector T cell response.

Antibodies to ICOS

Anti-ICOS antibodies may act as agonists of ICOS, mimicking and even surpassing the stimulatory effect of the native ICOS ligand on the receptor. Such agonism may result from ability of the antibody to promote multimerisation of ICOS on the T cell. One mechanism for this is where the antibodies form intercellular bridges between ICOS on the T cell surface and receptors on an adjacent cell (e.g., B cell, antigen-presenting cell, or other immune cell), such as Fc receptors and/or receptors to which the multi-specific antibody binds. Another mechanism is where antibodies having multiple (e.g., two) antigen-binding sites (e.g., two VH-VL domain pairs) bridge multiple ICOS receptor molecules and so promote multimerisation. A combination of these mechanisms may occur.

A bispecific antibody combining both ICOS agonism with PD-L1 antagonism may act via its PD-L1 binding arm (e.g., Fcab) to inhibit the negative co-regulatory signals generated by PD-L1 expressed on APCs, myeloid-derived suppressor cells (MDSC) or tumour cells, and may instead deliver a positive agonistic signal via its ICOS binding arm (e.g., Fab). See FIG. 1 .

An antibody to ICOS that acts to increase effector T cell activity represents a therapeutic approach in immunooncology and in other medical contexts where a CD8+ T cell response is beneficial. In many diseases and conditions involving an immune component, a balance exists between effector T cells (TEff) which exert the CD8+ T cell immune response, and regulatory T cells (TReg) which suppress that immune response by downregulating TEffs. The present invention relates to antibodies that modulate this TEff/TReg balance in favour of effector T cell activity. Antibodies that trigger the depletion of ICOS highly positive regulatory T cells would relieve the suppression of TEffs, and thus have a net effect of promoting the effector T cell response. An additional or complementary mechanism for an anti-ICOS antibody is via agonistic activity at the ICOS receptor level, to stimulate the effector T cell response.

The relative expression of ICOS on effector T cells (TEff) compared with regulatory T cells (TReg), and the relative activities of these cell populations, will influence the overall effect of an anti-ICOS antibody in vivo. An envisaged mode of action combines agonism of effector T cells with depletion of ICOS positive regulatory T cells. Differential and even opposing effects on these two different T cell populations may be achievable due to their different levels of ICOS expression. Dual-engineering of the variable and constant regions respectively of an anti-ICOS antibody can provide a molecule that exerts a net positive effect on effector T cell response by affecting the CD8/TReg ratio. An antigen-binding domain of an agonist antibody, which activates the ICOS receptor, may be combined with an antibody constant (Fc) region that promotes downregulation and/or clearance of highly expressing cells to which the antibody is bound. An effector positive constant region may be used to recruit cellular effector functions against the target cells (TRegs), e.g., to promote antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody dependent cell phagocytosis (ADCP). A bispecific antibody binding to ICOS and PD-L1 may trigger ADCC of PD-L1+ immunosuppressive cells (e.g., MDSC, tumour cells) and ICOS+ immunosuppressive cells (Tregs). The antibody may thus act both to promote effector T cell activation and to downregulate immunosuppressive T Regulatory cells. Since ICOS is more highly expressed on TRegs than on TEffs, a therapeutic balance may be achieved whereby Teff function is promoted while TRegs are depleted, resulting in a net increase in the T cell immune response (e.g, anti-tumour response or other therapeutically beneficial T cell response).

The ICOS binding site of multi-specific antibodies described herein may bind human ICOS. The antibodies target the ICOS extracellular domain and thereby bind to T cells expressing ICOS. Examples are provided of antibodies that have been designed to have an agonistic effect on ICOS, thus enhancing the function of effector T cells, as indicated by an ability to increase IFNγ expression and secretion. As noted, anti-ICOS antibodies may also be engineered to deplete cells to which they bind, which should have the effect of preferentially downregulating regulatory T cells, lifting the suppressive effect of these cells on the effector T cell response and thus promoting the effector T cell response overall. Regardless of their mechanism of action, it is demonstrated empirically herein that anti-ICOS antibodies stimulate T cell response and have anti-tumour effects in vivo, as shown in the Examples. Through selection of appropriate antibody formats such as those including constant regions with a desired level of Fc effector function, or absence of such effector function where appropriate, the anti-ICOS antibodies may be tailored for use in a variety of medical contexts including treatment of diseases and conditions in which an effector T cell response is beneficial and/or where suppression of regulatory T cells is desired.

A multispecific antibody may comprise a Fab having a VH-VL domain pair of any anti-ICOS antibody, e.g., any anti-ICOS antibody disclosed herein. The ICOS antibodies may be any of those described in PCT/GB2017/052352 (filed 9 Aug. 2017, published as WO2018/029474), the content of which is incorporated herein by reference. Exemplary antibodies include STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, the sequences of which are set out herein. Numerous further example anti-ICOS antibody sequences are also disclosed herein, including in the Tables. The Fab of the multispecific antibody in the present invention may comprise a VH domain and/or a VL domain having the CDRs of any such anti-ICOS antibody, and may comprise the VH and/or VL domain sequence of that antibody.

The anti-ICOS antibody may be one that competes for binding to human ICOS with an antibody (e.g., human IgG1, or an scFv) comprising the heavy and light chain complementarity determining regions (CDRs) of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, optionally an antibody comprising the VH and VL domains of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009.

The anti-ICOS antibody comprise one or more CDRs of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 (e.g., all 6 CDRs of any such antibody, or a set of HCDRs and/or LCDRs) or variants thereof as described herein.

The antibody may comprise an antibody VH domain comprising CDRs HCDR1, HCDR2 and HCDR3 and an antibody VL domain comprising CDRs LCDR1, LCDR2 and LCDR3, wherein the HCDR3 is an HCDR3 of an antibody selected from STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or comprises that HCDR3 with 1, 2, 3, 4 or 5 amino acid alterations. The HCDR2 may be the HCDR2 of the selected antibody or it may comprise that HCDR2 with 1, 2, 3, 4 or 5 amino acid alterations. The HCDR1 may be the HCDR1 of the selected antibody or it may comprise that HCDR1 with 1, 2, 3, 4 or 5 amino acid alterations.

The antibody may comprise an antibody VL domain comprising CDRs HCDR1, HCDR2 and HCDR3 and an antibody VL domain comprising CDRs LCDR1, LCDR2 and LCDR3, wherein the LCDR3 is an LCDR3 of an antibody selected from STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or comprises that LCDR3 with 1, 2, 3, 4 or 5 amino acid alterations. The LCDR2 may be the LCDR2 of the selected antibody or it may comprise that LCDR2 with 1, 2, 3, 4 or 5 amino acid alterations. The LCDR1 may be the LCDR1 of the selected antibody or it may comprise that LCDR1 with 1, 2, 3, 4 or 5 amino acid alterations.

An antibody may comprise:

-   -   an antibody VH domain comprising complementarity determining         regions HCDR1, HCDR2 and HCDR3, and     -   an antibody VL domain comprising complementarity determining         regions LCDR1, LCDR2 and LCDR3,     -   wherein the heavy chain complementarity determining regions are         those of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005,         STIM006, STIM007, STIM008 or STIM009 or comprise the STIM001,         STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006,         STIM007, STIM008 or STIM009 heavy chain complementarity         determining regions with 1, 2, 3, 4 or 5 amino acid alterations;         and/or     -   wherein the light chain complementarity determining regions are         those of antibody STIM001, STIM002, STIM002-B, STIM003, STIM004,         STIM005, STIM006, STIM007, STIM008 or STIM009, or comprise the         STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006,         STIM007, STIM008 or STIM009 light chain complementarity         determining regions with 1, 2, 3, 4 or 5 amino acid alterations.

An antibody may comprise a VH domain comprising a set of heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein

-   -   HCDR1 is the HCDR1 of STIM003,     -   HCDR2 is the HCDR2 of STIM003,     -   HCDR3 is the HCDR3 of STIM003,

or comprising that set of HCDRs with 1, 2, 3, 4, 5 or 6 amino acid alterations.

An antibody may comprise a VL domain comprising a set of light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein

-   -   LCDR1 is the LCDR1 of STIM003,     -   LCDR2 is the LCDR2 of STIM003,     -   LCDR3 is the LCDR3 of STIM003,

or comprising that set of LCDRs with 1, 2, 3 or 4 amino acid alterations.

Amino acid alterations (e.g., substitutions) may be at any residue position in the CDRs. Examples of amino acid alterations are those illustrated in FIG. 35 , FIG. 36 and FIG. 37 , which show alignments of variant sequences of anti-ICOS antibodies. Thus, an amino acid alteration in a STIM003 CDR may be a substitution of the residue present at the corresponding position in antibody CL-74570 or antibody CL-71642 as indicated in FIG. 36 .

Example amino acid alterations in STIM003 CDRs are substitutions at the following residue positions, defined according to IMGT:

-   -   In HCDR1, substitution at IMGT position 28, optionally a         conservative substitution, e.g., V28F.     -   In HCDR2, substitution at IMGT position 59, 63 and/or 64.         Optionally the substitution at position 59 is N591, the         substitution at position 63 is G63D and/or the substitution at         position 64 is D64N and/or D64S.     -   In HCDR3, substitution at IMGT position 106, 108, 109         and/or 112. Optionally the substitution at position 106 is         R106A, the substitution at position 108 is F108Y, the         substitution at position 109 is Y109F and/or the substitution at         position 112 is H112N. In LCDR1, substitution at position 36,         e.g., R36S.     -   In LCDR3, substitution at position 105, 108 and/or 109.         Optionally the substitution at position 105 is H105Q, the         substitution at position 108 is D108G and/or the substitution at         position 109 is M109N or M109S.

Antibodies of the invention may comprise VH and/or VL domain framework regions corresponding to human germline gene segment sequences. For example, it may comprise one or more framework regions of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009. The framework region or framework regions may be a FR1, FR2, FR3 and/or FR4.

An antibody of the invention may comprise an antibody VH domain which

-   -   (i) is derived from recombination of a human heavy chain V gene         segment, a human heavy chain D gene segment and a human heavy         chain J gene segment, wherein         -   the V segment is IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g.             V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g.,             V2-5*10);         -   the D gene segment is IGHD6-19 (e.g., IGHD6-19*01), IGHD3-10             (e.g., IGHD3-10*01) or IGHD3-9 (e.g., IGHD3-9*01); and/or         -   the J gene segment is IGHJ6 (e.g., IGHJ6*02), IGHJ4 (e.g.,             IGHJ4*02) or IGHJ3 (e.g., IGHJ3*02), or     -   (ii) comprises framework regions FR1, FR2, FR3 and FR4, wherein         -   FR1 aligns with human germline V gene segment IGHV1-18             (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g,             V3-11*01) or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2,             3, 4 or 5 amino acid alterations,         -   FR2 aligns with human germline V gene segment IGHV1-18             (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g,             V3-11*01) or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2,             3, 4 or 5 amino acid alterations,         -   FR3 aligns with human germline V gene segment IGHV1-18             (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g,             V3-11*01) or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2,             3, 4 or 5 amino acid alterations, and/or         -   FR4 aligns with human germline J gene segment IGJH6 (e.g.,             JH6*02), IGJH4 (e.g., JH4*02) or IGJH3 (e.g., JH3*02),             optionally with 1, 2, 3, 4 or 5 amino acid alterations.

FR1, FR2 and FR3 of the VH domain typically align with the same germline V gene segment. Thus, for example, the antibody may comprise a VH domain derived from recombination of human heavy chain V gene segment IGHV3-20 (e.g., VH3-20*d01), a human heavy chain D gene segment and a human heavy chain J gene segment IGJH4 (e.g., JH4*02). An antibody may comprise VH domain framework regions FR1, FR2, FR3 and FR4, wherein FR1, FR2 and FR3 each align with human germline V gene segment IGHV3-20 (e.g., IGVH3-20*d01) with up to 1, 2, 3, 4 or 5 amino acid alterations, and a FR4 that aligns with human germline J gene segment IGHJ4 (e.g., IGHJ4*02) with up to 1, 2, 3, 4 or 5 amino acid alterations. Alignment may be exact, but in some cases one or more residues can be mutated from germline, so there may be amino acid substitutions present, or in rarer cases deletions or insertions.

An antibody of the invention may comprise an antibody VL domain which

-   -   (i) is derived from recombination of a human light chain V gene         segment and a human light chain J gene segment, wherein         -   the V segment is IGKV2-28 (e.g., IGKV2-28*01), IGKV3-20             (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01) or             IGKV3-11 (e.g., IGKV3-11*01), and/or         -   the J gene segment is IGKJ4 (e.g., IGKJ4*01), IGKJ2 (e.g.,             IGKJ2*04), IGLJ3 (e.g., IGKJ3*01) or IGKJ1 (e.g., IGKJ1*01);             or     -   (ii) comprises framework regions FR1, FR2, FR3 and FR4, wherein         -   FR1 aligns with human germline V gene segment IGKV2-28             (e.g., IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39             (e.g., IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01),             optionally with 1, 2, 3, 4 or 5 amino acid alterations,         -   FR2 aligns with human germline V gene segment IGKV2-28             (e.g., IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39             (e.g., IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01),             optionally with 1, 2, 3, 4 or 5 amino acid alterations,         -   FR3 aligns with human germline V gene segment IGKV2-28             (e.g., IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39             (e.g., IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01),             optionally with 1, 2, 3, 4 or 5 amino acid alterations,             and/or         -   FR4 aligns with human germline J gene segment IGKJ4 (e.g.,             IGKJ4*01), IGKJ2 (e.g., IGKJ2*04), IGKJ3 (e.g., IGKJ3*01) or             IGKJ1 (e.g., IGKJ1*01), optionally with 1, 2, 3, 4 or 5             amino acid alterations.

FR1, FR2 and FR3 of the VL domain typically align with the same germline V gene segment. Thus, for example, the antibody may comprise a VL domain derived from recombination of human light chain V gene segment IGKV3-20 (e.g., IGKV3-20*01) and human light chain J gene segment IGKJ3 (e.g., IGKJ3*01). An antibody may comprise VL domain framework regions FR1, FR2, FR3 and FR4, wherein FR1, FR2 and FR3 each align with human germline V gene segment IGKV3-20 (e.g., IGKV3-20*01) with up to 1, 2, 3, 4 or 5 amino acid alterations, and a FR4 that aligns with human germline J gene segment IGKJ3 (e.g., IGKJ3*01) with up to 1, 2, 3, 4 or 5 amino acid alterations. Alignment may be exact, but in some cases one or more residues can be mutated from germline, so there may be amino acid substitutions present, or in rarer cases deletions or insertions.

An antibody according to the invention may comprise an antibody VH domain which is the VH domain of STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009, or which has an amino acid sequence at least 90 identical to the antibody VH domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009. The amino acid sequence identity may be at least 95%.

The antibody may comprise an antibody VL domain which is the VL domain of STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009, or which has an amino acid sequence at least 90% identical to the antibody VL domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009. The amino acid sequence identity may be at least 95%.

An antibody VH domain having the HCDRs of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or having a variant of those CDRs, may be paired with an antibody VL domain having the LCDRs of the same antibody, or having a variant of those CDRs. Similarly, the VH domain of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or a variant of that VH domain, may be paired with a VL domain of the same antibody, or a VL domain variant of the same antibody.

For instance, the antibody may comprise the antibody STIM001 VH domain and the STIM001 VL domain. In another example, the antibody may comprise the antibody STIM002 VH domain and the STIM002 VL domain. In another example, the antibody may comprise the antibody STIM003 VH domain and the STIM003 VL domain.

STIM001 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:366, comprising the CDRH1 amino acid sequence of Seq ID No:363, the CDRH2 amino acid sequence of Seq ID No:364, and the CDRH3 amino acid sequence of Seq ID No:365. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:367. STIM001 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:373, comprising the CDRL1 amino acid sequence of Seq ID No:370, the CDRL2 amino acid sequence of Seq ID No:371, and the CDRL3 amino acid sequence of Seq ID No:372. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:374. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:368 (heavy chain nucleic acid sequence Seq ID No:369). A full length light chain amino acid sequence is Seq ID No:375 (light chain nucleic acid sequence Seq ID No:376).

STIM002 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:380, comprising the CDRH1 amino acid sequence of Seq ID No:377, the CDRH2 amino acid sequence of Seq ID No:378, and the CDRH3 amino acid sequence of Seq ID No:379. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:381. STIM002 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:387, comprising the CDRL1 amino acid sequence of Seq ID No:384, the CDRL2 amino acid sequence of Seq ID No:385, and the CDRL3 amino acid sequence of Seq ID No:386. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:388 or Seq ID No:519. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:382 (heavy chain nucleic acid sequence Seq ID No:383). A full length light chain amino acid sequence is Seq ID No:389 (light chain nucleic acid sequence Seq ID No:390 or Seq ID NO:520).

STIM002-B has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:394, comprising the CDRH1 amino acid sequence of Seq ID No:391, the CDRH2 amino acid sequence of Seq ID No:392, and the CDRH3 amino acid sequence of Seq ID No:393. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:395. STIM002-B has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:401, comprising the CDRL1 amino acid sequence of Seq ID No:398, the CDRL2 amino acid sequence of Seq ID No:399, and the CDRL3 amino acid sequence of Seq ID No:400. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:402. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:396 (heavy chain nucleic acid sequence Seq ID No:397). A full length light chain amino acid sequence is Seq ID No:403 (light chain nucleic acid sequence Seq ID No:404).

STIM003 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:408, comprising the CDRH1 amino acid sequence of Seq ID No:405, the CDRH2 amino acid sequence of Seq ID No:406, and the CDRH3 amino acid sequence of Seq ID No:407. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:409 or Seq ID No:521. STIM003 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:415, comprising the CDRL1 amino acid sequence of Seq ID No:412, the CDRL2 amino acid sequence of Seq ID No:413, and the CDRL3 amino acid sequence of Seq ID No:414. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:4416. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:410 (heavy chain nucleic acid sequence Seq ID No:411 or Seq ID No:522). A full length light chain amino acid sequence is Seq ID No:417 (light chain nucleic acid sequence Seq ID No:418).

STIM004 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:422, comprising the CDRH1 amino acid sequence of Seq ID No:419, the CDRH2 amino acid sequence of Seq ID No:420, and the CDRH3 amino acid sequence of Seq ID No:421. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:423. STIM004 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:429, comprising the CDRL1 amino acid sequence of Seq ID No:426, the CDRL2 amino acid sequence of Seq ID No:427, and the CDRL3 amino acid sequence of Seq ID No:428. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:430 or Seq ID No:431. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:424 (heavy chain nucleic acid sequence Seq ID No:425). A full length light chain amino acid sequence is Seq ID No:432 (light chain nucleic acid sequence Seq ID No:433 or Seq ID no: 434).

STIM005 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:438, comprising the CDRH1 amino acid sequence of Seq ID No:435, the CDRH2 amino acid sequence of Seq ID No:436, and the CDRH3 amino acid sequence of Seq ID No:437. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:439. STIM005 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:445, comprising the CDRL1 amino acid sequence of Seq ID No:442, the CDRL2 amino acid sequence of Seq ID No:443, and the CDRL3 amino acid sequence of Seq ID No:444. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:446. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:440 (heavy chain nucleic acid sequence Seq ID No:441). A full length light chain amino acid sequence is Seq ID No:447 (light chain nucleic acid sequence Seq ID No:448).

STIM006 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:452, comprising the CDRH1 amino acid sequence of Seq ID No:449, the CDRH2 amino acid sequence of Seq ID No:450, and the CDRH3 amino acid sequence of Seq ID No:451. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:453. STIM006 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:459, comprising the CDRL1 amino acid sequence of Seq ID No:456, the CDRL2 amino acid sequence of Seq ID No:457, and the CDRL3 amino acid sequence of Seq ID No:458. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:460. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:454 (heavy chain nucleic acid sequence Seq ID No:455). A full length light chain amino acid sequence is Seq ID No:461 (light chain nucleic acid sequence Seq ID No:462).

STIM007 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:466, comprising the CDRH1 amino acid sequence of Seq ID No:463, the CDRH2 amino acid sequence of Seq ID No:464, and the CDRH3 amino acid sequence of Seq ID No:465. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:467. STIM007 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:473, comprising the CDRL1 amino acid sequence of Seq ID No:470, the CDRL2 amino acid sequence of Seq ID No:471, and the CDRL3 amino acid sequence of Seq ID No:472. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:474. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:468 (heavy chain nucleic acid sequence Seq ID No:469). A full length light chain amino acid sequence is Seq ID No:475 (light chain nucleic acid sequence Seq ID No:476).

STIM008 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:480, comprising the CDRH1 amino acid sequence of Seq ID No:477, the CDRH2 amino acid sequence of Seq ID No:478, and the CDRH3 amino acid sequence of Seq ID No:479. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:481. STIM008 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:487, comprising the CDRL1 amino acid sequence of Seq ID No:484, the CDRL2 amino acid sequence of Seq ID No:485, and the CDRL3 amino acid sequence of Seq ID No:486. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:488. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:482 (heavy chain nucleic acid sequence Seq ID No:483). A full length light chain amino acid sequence is Seq ID No:489 (light chain nucleic acid sequence Seq ID No:490).

STIM009 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:494, comprising the CDRH1 amino acid sequence of Seq ID No:491, the CDRH2 amino acid sequence of Seq ID No:492, and the CDRH3 amino acid sequence of Seq ID No:493. The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:495. STIM009 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:501, comprising the CDRL1 amino acid sequence of Seq ID No:498, the CDRL2 amino acid sequence of Seq ID No:499, and the CDRL3 amino acid sequence of Seq ID No:500. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:502. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:496 (heavy chain nucleic acid sequence Seq ID No:497). A full length light chain amino acid sequence is Seq ID No:503 (light chain nucleic acid sequence Seq ID No:504).

The sequences of these and other anti-ICOS antibodies are shown in the Tables. Antibodies STIM001-009 are described in more detail in PCT/GB2017/052352 (WO2018/029474) filed 9 Aug. 2017.

Further exemplary anti-ICOS antibodies include those described in GB patent application no 1721338.0 filed 19 Dec. 2017 and the corresponding international patent application claiming priority from it and filed on 19 Dec. 2018 entitled “Antibodies to ICOS”. An antibody according to the present invention may comprise an anti-ICOS Fab in which the VH and/or VL domain is the VH and/or VL domain of any anti-ICOS antibody described therein, including any of STIM017, STIM020, STIM021, STIM022, STIM023, STIM039, STIM040, STIM041, STIM042, STIM043, STIM044, STIM050, STIM051, STIM052, STIM053, STIM054, STIM055, STIM056, STIM057, STIM058, STIM059, STIM060, STIM061, STIM062, STIM063, STIM064, STIM065 and STIM066, or a variant thereof. The VH and VL domain sequences of these antibodies are incorporated herein by reference.

In further embodiments, the VH and/or VL domain of the anti-ICOS Fab may be any VH and/or VL domain:

-   -   a. of the antibody 7F12, 37A10, 35A9, 36E10, 16G10, 37A10S713,         37A10S714, 37A10S715, 37A10S716, 37A10S717, 37A10S718, 16G10S71,         16G10S72, 16G10S73, 16G10S83, 35A9S79, 35A9S710, 35A9S89 or any         other antibody described in WO2016/154177 and US2016/0304610;     -   b. of the antibody 422.2, H2L5, or any other antibody described         in WO2016/120789 and US2016/0215059;     -   c. of the antibody 314-8, the antibody produced from hybridoma         CNCM I-4180, or any other antibody described in WO2014/033327         and US2015/0239978;     -   d. of the antibody Icos145-1, the antibody produced by hybridoma         CNCM I-4179, or any other antibody described in WO2012/131004,         U.S. Pat. No. 9,376,493 and US2016/0264666;     -   e. of the antibody JMAb 136, “136”, or any other antibody         described in WO2010/056804;     -   f. of the antibody MIC-944, 9F3 or any other antibody described         in WO99/15553, U.S. Pat. Nos. 7,259,247, 7,132,099, 7,125,551,         7,306,800, 7,722,872, WO05/103086, U.S. Pat. Nos. 8,318,905 and         8,916,155;     -   g. of any JMAb antibody, e.g., any of JMAb-124, JMAb-126,         JMAb-127, JMAb-128, JMAb-135, JMAb-136, JMAb-137, JMAb-138,         JMAb-139, JMAb-140, JMAb-141, e.g., JMAb136, or any other         antibody described in WO98/3821, U.S. Pat. No. 7,932,358B2,         US2002/156242, U.S. Pat. Nos. 7,030,225, 7,045,615, 7,279,560,         7,226,909, 7,196,175, 7,932,358, 8,389,690, WO02/070010, U.S.         Pat. Nos. 7,438,905, 7,438,905, WO01/87981, U.S. Pat. Nos.         6,803,039, 7,166,283, 7,988,965, WO01/15732, U.S. Pat. Nos.         7,465,445 and 7,998,478;     -   h. of the antibody 17G9 or any other antibody described in         WO2014/08911;     -   i. of any antibody described in WO2012/174338;     -   j. of any antibody described in US2016/0145344;     -   k. of any antibody described in WO2011/020024, US2016/002336,         US2016/024211 and U.S. Pat. No. 8,840,889; or     -   l. of any antibody described in U.S. Pat. No. 8,497,244;     -   m. of the antibody known as GSK3359609;     -   n. of the antibody known as JTX-2011;     -   o. of antibody clone ISA-3 (eBioscience), clone SP98 (Novus         Biologicals), clone 1 G1, clone 3G4 (Abnova Corporation), clone         669222 (R&D Systems), clone TQ09 (Creative Diagnostics), or         clone C398.4A (BioLegend); or     -   p. of the hamster antibody C398.4 or a humanised antibody         thereof as described in WO2018/187613 or US2018/0289790, e.g.,         ICOS.33 IgG1f S267E. Other examples include ICOS.4, ICOS34 G1f,         ICOS35 G1f, 17C4, 9D5, 3E8, 1D7a, 1D7b and 2644. The antibody         sequences are summarised in Table 35 therein;

The VH and VL domain sequences of these antibodies are incorporated herein by reference. An anti-ICOS Fab of the present invention may comprise the CDRs of any such VH and/or VL domain.

Targeting Other Antigens

A multispecific antibody may bind further target antigens in addition to ICOS and PD-L1 and/or it may be combined with one or more other antibodies that bind further target antigens. Optionally, the multispecific antibody comprises two Fabs, wherein one Fab comprises a binding site for ICOS and wherein the other Fab comprises a binding site for another target antigen.

The further target antigen may be selected from immune checkpoint inhibitors (such as PD-1, CTLA-4, TIGIT, TIM-3, LAG-3 and VISTA, e.g. TIGIT, TIM-3 and LAG-3), immune modulators (such as BTLA, hHVEM, CSF1R, CCR4, CD39, CD40, CD73, CD96, CXCR2, CXCR4, CD200, GARP, SIRPα, CXCL9, CXCL10, CXCL11 and CD155, e.g. GARP, SIRPα, CXCR4, BTLA, hVEM and CSF1R), immune activators (such as CD137, GITR, OX40, CD40, CXCR3 (e.g. agonistic anti-CXCR3 antibodies), CD27, and CD3.

The second Fab of the multispecific antibody, or the additional antibody that is used in combination with the multispecific antibody, may alternatively comprise a binding site for PD-L1, and a number of exemplary anti-PD-L1 antibodies are described herein.

In one embodiment, the PD-L1 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from any one of the anti-PD-L1 antibodies selected from atezolizumab (Roche), avelumab (Merck), BMS-936559/MDX-1105 (BMS), durvalumab/Medi4736 (Medimmune), KN-035, CA-170, FAZ-053, M7824, ABBV-368, LY-3300054, GNS-1480, YW243.55.S70, REGN3504 and any of the PD-L1 antibodies disclosed in WO2017/034916, WO2017/020291, WO2017/020858, WO2017/020801, WO2016/111645, WO2016/197367, WO2016/061142, WO2016/149201, WO2016/000619, WO2016/160792, WO2016/022630, WO2016/007235, WO2015/179654, WO2015/173267, WO2015/181342, WO2015/109124, WO2015/112805, WO2015/061668, WO2014/159562, WO2014/165082, WO2014/100079, WO2014/055897, WO2013/181634, WO2013/173223, WO2013/079174, WO2012/145493, WO2011/066389, WO2010/077634, WO2010/036959, WO2010/089411 or WO2007/005874, which antibodies and sequences are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds PD-L1, e.g. hPD-L1. In one embodiment, the PD-L1 antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from any one of the anti-PD-L1 antibodies selected from atezolizumab/MPDL3280A (Roche), avelumab/MSB0010718C (Merck), BMS-936559/MDX-1105 (BMS), durvalumab/Medi4736 (Medimmune), KN-035, CA-170, FAZ-053 M7824, ABBV-368, LY-3300054, GNS-1480, YW243.55.S70, REGN3504 and any of the PD-L1 antibodies disclosed in WO2017/034916, WO2017/020291, WO2017/020858, WO2017/020801, WO2016/111645, WO2016/050721, WO2016/197367, WO2016/061142, WO2016/149201, WO2016/000619, WO2016/160792, WO2016/022630, WO2016/007235, WO2015/179654, WO2015/173267, WO2015/181342, WO2015/109124, WO2015/195163, WO2015/112805, WO2015/061668, WO2014/159562, WO2014/165082, WO2014/100079, WO2014/055897, WO2013/181634, WO2013/173223, WO2013/079174, WO2012/145493, WO2011/066389, WO2010/077634, WO2010/036959, WO2010/089411 or WO2007/005874, which antibodies and sequences are incorporated herein by reference.

In one embodiment, the another target antigen is CTLA-4. In one embodiment, the another target antigen is TIGIT. In one embodiment, the another target antigen is TIM-3. In one embodiment, the another target antigen is LAG-3. In one embodiment, the another target antigen is GITR. In one embodiment, the another target antigen is VISTA. In one embodiment, the another target antigen is CD137. In one embodiment, the another target antigen is SIRPα. In one embodiment, the another target antigen is CXCL10. In one embodiment, the another target antigen is CD155. In one embodiment, the another target antigen is CD40.

In another embodiment, the multispecific antibody binds another target antigen which is PD-1 and the binding to PD-1 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CTLA4 and the binding to CTLA4 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is TIGIT and the binding to TIGIT is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is TIM-3 and the binding to TIM-3 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is LAG3 and the binding to LAG3 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is VISTA and the binding to VISTA is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is BTLA and the binding to BTLA is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is hHVEM and the binding to hHVEM is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CSF1R and the binding to CSF1R is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CCR4 and the binding to CCR4 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD39 and the binding to CD39 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD40 and the binding to CD40 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD73 and the binding to CD73 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD96 and the binding to CD96 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CXCR2 and the binding to CXCR2 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CXCR4 and the binding to CXCR4 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD200 and the binding to CD200 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is GARP and the binding to GARP is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is SIRPα and the binding to SIRPα is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CXCL9 and the binding to CXCL9 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CXCL10 and the binding to CXCL10 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CXCL11 and the binding to CXCL11 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD155 and the binding to CD155 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD137 and the binding to CD137 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is GITR and the binding to GITR is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is OX40 and the binding to OX40 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD40 and the binding to CD40 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CXCR3 and the binding to CXCR3 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD27 and the binding to CD27 is provided by an antigen-binding domain (for example, a V_(H), a V_(L) or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

In another embodiment, the multispecific antibody binds another target antigen which is CD3 and the binding to CD3 is provided by an antigen-binding domain (for example, a V_(H), a VL or a paired V_(H) and V_(L)) having any of the sequences, including CDR sequences (for example CDRH3 and/or CDRL3) or variable region sequences as described in Aspect 1A hereinbelow.

As an alternative to providing the binding site for the further target antigen as part of the multispecific antibody, the multispecific antibody may instead be used in combination with an additional binding molecule (e.g., antibody) comprising an antigen-binding domain to the further target antigen, including any of the example antigen-binding domains mentioned.

In any of the following embodiments, a particular antigen-binding site specifically binds to a human target. In one embodiment, the antigen-binding site specifically binds an immune checkpoint inhibitor. In one embodiment, the antigen-binding site specifically binds an immune checkpoint inhibitor selected from PD-1, CTLA-4, TIGIT, TIM-3, LAG-3 and VISTA. In one embodiment, the antigen-binding site specifically binds an immune checkpoint inhibitor selected from TIGIT, CTLA-4, TIM-3 and LAG-3.

In one embodiment, the antigen-binding site specifically binds PD-1, e.g. human PD-1. In one embodiment, the PD-1 antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the VH, or the VL or the VH and VL region from pembrolizumab (Keytruda®/MK-3475), nivolumab (Opdivo®/BMS-936558/MDX-1106), MEDI-0680/AMP514, PDR001, Lambrolizumab, BMS-936558, REGN2810, BGB-A317, BGB-108, PDR-001, SHR-1210, JS-001, JNJ-63723283, AGEN-2034, PF-06801591, genolimzumab, MGA-012, IBI-308, BCD-100, TSR-042 ANA011, AUNP-12, KD033, MCLA-134, mDX400, muDX400, STI-A1110, AB011, 244C8, 388D4, XCE853, or pidilizumab/CT-011, or from any one of the anti-PD-1 antibodies described in WO2015/112800 & US2015/0203579 (including the antibodies in Tables 1 to 3), U.S. Pat. Nos. 9,394,365, 5,897,862 and 7,488,802, WO2017/087599 (including antibody SSI-361 and SHB-617), WO2017/079112, WO2017/071625 (including deposit C2015132, hybridoma LT004, and antibodies 6F5/6 F5 (Re), 6F5H1 L1 and 6F5 H2L2), WO2017/058859 (including PD1AB-1 to PD1AB-6), WO2017/058115 (including 67D9, c67D9, and hu67D9), WO2017/055547 (including 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375 and 13112.15380), WO2017/040790 (including AGEN2033w, AGEN2034w, AGEN2046w, AGEN2047w, AGEN2001w and AGEN2002w), WO2017/025051 & WO2017/024515 (including 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb and 1.153.7 hAb), WO2017/025016 & WO2017/024465 (including antibody A to antibody I), WO2017/020858 & WO2017/020291 (including 1.4.1, 1.14.4, 1.20.15 and 1.46.11), WO2017/019896 & WO2015/112900 & US2015/0210769 (including BAP049-hum01 to BAP049-hum16 and BAP049-Clone-A to BAP049-Clone-E), WO2017/019846 (including PD-1 mAb 1 to PD-1 mAb 15), WO2017/016497 (including MHC723, MHC724, MHC725, MHC728, MHC729, m136-M13, m136-M19, m245-M3, m245-M5 and m136-M14), WO2016/201051 (including antibody EH12.2H7, antibody hPD-1 mAb2, antibody hPD-1 mAb7, antibody hPD-1 mAb9, antibody hPD-1 mAb15, or an anti-PD-1 antibody selected from Table 1), WO2016/197497 (including DFPD1-1 to DFPD1-13), WO2016/197367 (including 2.74.15 and 2.74.15.hAb4 to 2.74.15.hAb8), WO2016/196173 (including the antibodies in Table 5, and FIGS. 1-5 ), WO2016/127179 (including R3A1, R3A2, R4B3, and R3D6), WO2016/077397 (including the antibodies described in Table 1 of Example 9), WO2016/106159 (including the murine antibodies in Table 3 of Example 2 and the humanised antibodies in Tables 7, 8 and 9 of Example 3), WO2016/092419 (including C1, C2, C3, EH12.1, mAb7-G4, mAb15-G4, mAb-AAA, mAb15-AAA), WO2016/068801 (including clone A3 and its variants and the other antibodies described in FIGS. 1 to 4 ), WO2016/014688 (including 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, 7A4, and 7A4D and the humanised antibodies of Examples 9/10), WO2016/015685 (including 10F8, BA08-1, BA-08-2 and 15H6), WO2015/091911 & WO2015/091910 (including the anti-canine PD-1 antibodies in Examples 2, 3 and 4), WO2015/091914 (including the anti-canine PD-1 antibodies in Table 3), WO2015/085847 (including mAb005, H005-1 to H005-4), WO2015/058573 (including cAB7), WO2015/036394 (including LOPD180), WO2015/035606 (including the antibodies in Table 1 of Example 2, in Tables 14, 15 and 16 of Example 7 and in tables 20, 21 and 22 of Example 11), WO2014/194302 (including GA2, RG1B3, RG1H10, RG2A7, RG2H10, SH-A4, RG4A6, GA1, GB1, GB6, GH1, A2, C7, H7, SH-A4, SH-A9, RG1H11, and RG6B), WO2014/179664 (including 9A2, 10B11, 6E9, APE1922, APE1923, APE1924, APE1950, APE1963 and APE2058), WO2014/206107 (including clone 1, 10, 11, 55, 64, 38, 39, 41 and 48), WO2012/135408 (including h409A11, h409A16, and h409A17), WO2012/145493 (including antibodies 1E3, 1E8, 1H3 and h1H3 Var 1 to h1H3 Var 14), WO2011/110621 (including antibody 949 and the modified versions disclosed in FIGS. 1 to 11 ), WO2011/110604 (including antibody 948 and the modified versions disclosed in FIGS. 3 to 11 ), WO2010/089411 (including CNCM deposit number 1-4122, 1-4080 or 1-4081), WO2010/036959 (including the antibodies in Table 1 of Example 1), WO2010/029435 & WO2010/029434 (including clones 2, 10 and 19), WO2008/156712 (including hPD-1.08A, hPD-1.09A, h409A11, h409A16 and h409A17 and the antibodies described in Example 2, Table H, Example 4 and table IV), WO2006/121168 (including clones 17D8, 4H1, 5C4, 4A11, 7D3, 5F4, and 2D3), WO2004/004771 or WO2004/056875 (including PD1-17, PD1-28, PD1-33, PD1-35, PD1-F2 and the Abs described in Table 1); the sequences and features of the anti-PD-1 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds TIGIT, e.g. human TIGIT. In one embodiment, the TIGIT antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the VH, or the VL or the VH and VL region from RG-6058 (MTIG-7192A) or from any one of the anti-TIGIT antibodies described in WO2017/053748 (including 1A4, 1D3, 4A3, 10A7, 4.1D3.Q1E, h10A7.K4G3, 4.1D3 and the other antibodies described in Examples 1 and 2), WO2017/037707 (including VSIG9 #1 and 258-csl #4), WO2017/030823 (including 14D7, 26610 and humanized versions in Example 3), WO2016/191643 (including 313R11, 313R12, 313R14, 313R19, 313R20, ATCC PTA-122180 and ATCC PTA-122181), WO2016/106302 (including 1462, 13E6, 6F9, 11G11, 10C9, 16F6, 11C9, 27A9, 10D7, 20G6, 24E8, 24G1, 27F1, 15A6, 4E4, 13D1, 91311, 1068, 22G2, 19H2, 8C8, 17G4, 25E7, 26D8 and 16A8), WO2016/028656 (including 14A6, 28H5 or 3106 and humanized versions from Example 6), and WO2009/126688 (US2013/0251720, including 10A7 and 1F4); the sequences and features of the anti-TIGIT antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds TIM-3, e.g. human TIM-3. In one embodiment, the TIM-3 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the VH, or the VL or the VH and VL region from F38-2E2 (BioLegend), clone 2E2 (Merck Millipore), clone 6136E2, clone 024 (Sino Biological) clone 344801 (R&D Systems), clone E-18, clone H-191 (Santa Cruz Biotechnology), or clone 13A224 (United States Biological), TSR-022 (Tesaro) or from any one of the anti-TIM-3 antibodies described in WO2017/079115 (including anti-TIM3 antibodies listed in tables 30-38), WO2017/055404 (including PD1TIM3-0389, PD1TIM3-0168, PD1TIM3-0166, TIM3-0038, TIM3-0018, TIM3-0028, TIM3-0438—Table C), WO2017/031242 (Table 10), WO2016/179194 (including antibodies in FIG. 1 b , including mAb F38-2E2 and 2E2), WO2016/171722 (including 344823 and antibodies from the hybridomas 7D11, 10G12, 11G8, 8B.2C12 and 25F.1D6), WO2016/161270 (including APE5137 and APE5121), WO2016/111947 (including mAb5, mAb13, mAb15, mAb17, mAb21, mAb22, mAb26, mAb27, mAb48, mAb58 and mAb91), WO2016/071448 (including TIM3-0016, TIM3-0018, TIM3-0021, TIM3-0022, TIM3-0026, TIM3-0028, TIM3-0030, TIM3-0033, TIM3-0038, TIM3-0433, TIM3-0434, TIM3-0438 and TIM3-0443), WO2016/068802 (including 1B9, 1H9, 1H10, 2C7, 2F4, 2G6, 1D9, 1F4 and 2C8—FIGS. 1, 2 & 3 ), WO2016/068803 (including A3,1310, G6, G7, G9, A11 and A11_gl—FIGS. 1, 2 & 3 ), WO2015/117002 (including ABTIM3, ABTIM3-hum02, ABTIM3-hum05, ABTIM3-hum06, ABTIM3-hum09, ABTIM3-hum10, ABTIM3-hum12, ABTIM-hum01, ABTIM-hum04, ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum04, ABTIM3-hum21, ABTIM3-hum03, ABTIM3-hum11 and antibodies listed in Table 9), WO2015/048312 (including 5D12), WO2014/022332 (including 2C12), WO2013/006490 (including antibodies in Table 1), WO2011/155607 (including 512, 644, 4545, 4177, 8213, 344823 and 34823), WO2003/063792 (including antibody 8B.2C12 and 25F.1D6), WO2017/019897 (including antibody molecules disclosed in Tables 1-4, including ABTIM3, ABTIM3-hum20, ABTIM3-hum22 and ABTIM3-hum23), WO2016/079050 & WO2016/079050 (including Tim3_0022, Tim3_0016, Tim3_0018, Tim3_00122, Tim3_0022, Tim3_0021, Tim3_0028, Tim3_0026, Tim3_0033, Tim3_0038, Tim3_0030, 1.7. E10, F38-2EL and 27-12E12); the sequences and features of the anti-TIM-3 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds LAG-3, e.g. human LAG-3. In one embodiment, the LAG-3 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from antibody clone 1764 (Enzo Life Sciences), or clone 333210 (R&D Systems), or clone 14L676 (United States Biological), or C9B7W (PharMingen), or 11E, or IMO321, or mAb C9B7W (BioXcell) or from any one of the anti-LAG-3 antibodies described in WO95/30750, WO2004/078928, WO2008/132601 (including IMP731 Lag-3 Ab, IMP321, A9H12 Lag-3 mAb and 31G11), WO2010/019570 (including 25F7, 26H10, 25E3, 8B7, 11F2 and 17E5), WO2014/140180 (including H5L7, H5L7BW, IMP731 and antibodies in Tables 3 & Table 7), WO2014/179664 (including APE03109), WO2014/008218 (including Lag3.1, Lag3.5, Lag3.6, Lag3.7 and Lag3.8), WO2015/042246, WO2015/116539 (including BMS-986016), WO2015/138920 (including BAP050-hum01 to BAP050-hum20, huBAP050(Ser), BAP050-hum01-Ser to BAP050-hum20-Ser, BAP050-Clone-F, BAP050-Clone-G, BAP050-Clone-H, BAP050-Clone-I, BAP050-Clone-J, BAP050 and BAP050-chi), WO2015/198312, WO2016/028672 (including Ab1, Ab2, Ab3, Ab4, Abs, Ab6, Ab7, Ab8 and Ab9), WO2016/126858, WO2016/200782 (including LAG-3 mAb1 to LAG-3 mAb6), WO2017/015560 (including L32D10, L3E3, L3C5, L35D4, L35G6, L33H11, L32A9, L32A4, L3A1 and the antibodies listed in Table 3), WO2017/062888 (including mAb1, H4H15477P, H4H15483P, H4H15484P, H4H15491, H4H17823P, H4H17826P2, H4H17828P2, H4sH15460P, H4sH15462P, H4sH15463P, H4sH15464P, H4sH15466P, H4sH15467P, H4sH15470P, H4sH15475P, H4sH15479P, H4sH15480P, H4sH15482P, H4sH15488P, H4sH15496P2, H4sH15498P2, H4sH15505P2, H4sH15518P2, H4sH15523P2, H4sH15530P2, H4sH15555P2, H4sH15558P2, H4sH15567P2 and H4H17819P), WO2017/019894, WO2017/037203 (including 8E2, 13E2, 34F4, 1764 and IMP761), WO2017/087589 (including 11B09) or WO2017/087901; the sequences and features of the anti-LAG-3 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds VISTA, e.g. human VISTA. In one embodiment, the VISTA antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from any one of the anti-VISTA antibodies described in WO2016/207717 & WO2015/097536 (including VSTB50, VSTB53, VSTB60, VSTB95, VSTB112, VSTB116, VSTB174, VSTB175, VSTB149, VSTB140 and the antibodies in Table 1A and Examples 7 and 8) and WO2014/190356 (including clone 2D3 and 18C3); the sequences and features of the anti-VISTA antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CTLA-4, e.g. hCTLA-4. In one embodiment, the CTLA-4 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from ipilimumab (MDX-010, CAS No. 477202-00-9), tremelimumab (ticilimumab/CP-675,206), antibody clone 2F1, clone 1F4 (Abnova Corporation), clone 9H10 (EMD Millipore), clone BNU3 (GeneTex), clone 1 E2, clone AS32 (Lifespan Biosciences) clone A3.4H2.H12 (Acris Antibodies), clone 060 (Sino Biological), clone BU5G3 (Creative Diagnostics), clone MIH8 (MBL International), clone A3.6B10.G1, or clone L3D10 (BioLegend) or from any one of the anti-CTLA-4 antibodies described in WO2017/087588 (ISVs disclosed in FIG. 2 ), WO2017/084078 (clones C2, C4, C10, C11, C12 and C13, and FIGS. 4-7 ), WO2016/196237 (including AGEN1884w, AGEN2041w, the sequences in FIGS. 19A, 19B and Tables 1-6), WO2016/130986 & WO2016/130898 (including E8, F7 and the Abs described in Table 4), WO2016/015675 (including hybridoma LT001 and antibodies 8D2, 8D2H1L1, 8D2H2L2, 8D2H3L3, 8D2H2L15 and 8D2H2L17), WO2012/120125 (including 3610, 8H5, and the Abs identified in Examples 1, 2, 3 and 5), WO2010/097597 (including JMW-3B3 and the variants and fragments disclosed), WO2009/100140 (including 10D1, 1H5, 3A4, 6C10 and the antibodies described in FIGS. 1 to 6 ), WO2007/008463 & WO2006/101692 & WO2006/101691 & WO2006/048749 & WO2005/09238, (including 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, 12.9.1.1, and 10D1), WO2006/096491 (including ATCC Deposit No. 11.2.1 11.2.1.4 PTA-5169 and 4.1.1 4.1.1.1 PTA-5166), WO2006/066568 (including TGN2122.C, TGN2422.C, 4.8H10H5 and 4.3F6B5 and the antibodies described in tables 3 to 14), WO2006/029219 (including L3D10, L1B11, K4G4, KM10, and YL2), WO2004/029069 (including ATCC deposit number PTA-4537), WO01/54732 (including antibodies 25, 26, 27, 29, 33, 34, 35, 36 and 38), WO01/14424 (including 3A4, 9A5, 2E2, 2E7, 4B6, 4E10, 5C4, 5G1, 11E8, and 11G1 and the antibodies identified in Examples 3 and 4 and table 3) and WO00/37504 (including 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1); the sequences and features of the anti-CTLA-4 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds an immune modulator. In one embodiment, the antigen-binding site specifically binds an immune modulator selected from BTLA, hHVEM, CSF1R, CCR4, CD39, CD40, CD73, CD96, CXCR2, CXCR4, CD200, GARP, SIRPα, CXCL9, CXCL10, CXCL11 and CD155, or from BTLA, hHVEM, CSF1R, CCR4, CD39, CD40, CD73, CD96, CXCR2, CXCR4, CD200, GARP, SIRPα, CXCL9, CXCL10 and CD155. In one embodiment, the antigen-binding site specifically binds an immune modulator selected from GARP, SIRPα, CXCR4, BTLA, hVEM and CSF1R.

In one embodiment, the antigen-binding site specifically binds GARP, e.g. human GARP. In one embodiment, the GARP antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from G14D9, Plato-1, 272, G6, 50 G10 or 7B11 or from any one of the anti-GARP antibodies described in WO2007/113301 & WO2015/015003 (including MHGARP8, LHG-10, LHG-10-D, LHG-10.3-D, LHG-10.4-D, LHG-10.5-D, LHG-10.6-D, LHG-10.3, LHG-10.4, LHG-10.5, LHG-10.6, 27E10, MHGARP1, MHGARP2, MHGARP3, MHGARP4, MHGARP5, MHGARP6, MHGARP7 and MHGARP9), WO2017/051888 (including 110F, 105F, c151D, c198D, h198D, h151D, h151D-H1L1 and h198D-H3L4); the sequences and features of the anti-GARP antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds SIRPα, e.g. human SIRP□. In one embodiment, the SIRPα antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the VH, or the VL or the VH and VL region from ED9 (ThermoFisher), or 602411 (Novus Biologicals), or from any one of the anti-SIRPα, antibodies described in WO97/48723, WO00/24869 (including 10C4), WO00/66159 (including ED9 and ED17), WO01/40307, WO02/092784 (including SE5A5, SE7C2 and SE12C3), WO2004/108923 (including SE12C3 and 2F34), WO2009/046541 (including P84), WO2011/076781, WO2012/172521, WO2012/040207 (including SE5A5 and mouse P84), WO2013/056352 (including 29-AM4-5, Ab AM4-5, AM5-1, AM5-3, AM5-5, AM5-6, SIRPalpha-AM3-35, AM4-1, SIRP29-AM3-35, SIRP29-AM4-5, SIRP29-AM4-1, 29-AM2-2, 29-AM4-4, 29-AM4-1, 29-AM4-5, 29-AM3-35 and SIRP29-AM3-63), WO2016/063233, WO2016/205042 (including P362) or WO2015/138600 (including KWAR23); the sequences and features of the anti-SIRPα antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CXCR4, e.g. human CXCR4. In one embodiment, the CXCR4 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region of ulocuplumab/BMS-936564, clone 44717.111 or PF-06747143 or from any one of the anti-CXCR4 antibodies described in WO97/49424 (including MAB12G5), WO99/50461, WO01/42308, WO03/066830 & WO2003/066830 (including Ab124 and Ab125), WO2004/059285 (including ALX40-4C), WO2006/089141 (including mAbs 2N, 6R, 18, 19, 20, 33 and 48), WO2007/005605, WO2008/142303 (including MAB170, MAB171, MAB173 and MAB172), WO2008/060367 & WO2013/071068 & WO2015/015401 (including BMS-936564/MDX-1338), WO2009/140124 (including antibody I, II, III, IV and V), WO2009/117706 (including 701, 708, 716, 717, 718 and 4G10), WO2011/161266 (including 4CXCR100, 4CXCR103, 4CXCR104, 4CXCR101, 4CXCR238D2 and 4CXCR238D4), WO2011/098762 (including C-9P21 (Table 1), B-1M22 (Table 2), C1124 (Table 3), D-1K21 (Table 4) and 9N10 (Table 5)), WO2012/175576, WO2013/013025 (including 2A4, 6C7, 4C1, 7C8, 5C9 and 5E1), WO2013/017566 (including Mab 427aB1 and 515H7), WO2013/017562 (including 1-3859 Mab and 515H7), WO2015/069874 (including antibodies corresponding to Seq ID numbers 25 and 29), WO2015/015401 (including 12A11, 6B6, 3G10, m3G10.hIgG1, m3G10.hIgG4, h3G10.A57.hIgG1, h3G10.A57.A58A.hIgG1, h3G10.1.91.A58A.hIgG1, h3G10.1.91.A58B.hIgG1 and h3G10.2.37.2.72.hIgG1), WO2016/156570 (including 281F12, 281A6 and 281D4), WO2016/109872 (including antibodies listed in tables 1, 2, 9 & 12, M3-114-6H, AM4-272-6H, AM3-523-6H, AM4-272, AM3-114, AM3-523, AM4-746 and AM4-1121), WO2017/071625, WO2012/175576, WO2010/125162 & WO2012/055980 & WO2011/121040 & WO2010/037831 (including c414H5 (414H5), c515H7 (515H7) and 301aE5), WO2009/138519 (including ALX40-4C, 238D2, 238D4, 23765 antibodies and sequences listed in table 1, table 1.1, table A-I, table B-1.1 & B-5), WO2011/042398 (including 238D2 and 238D4), WO2011/083140 (including those disclosed in Tables C-2, C-3, C-4 & C-5, FIG. 2 and ALX-0651, 15H3, 10E12, 10G10, 23866, 10E9, 281E10, 10A10, 14A2 and 15A1) or WO2011/083141); the sequences and features of the anti-CXCR4 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds BTLA, e.g. hBTLA. In one embodiment, the BTLA antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from antibody clone 1B7, clone 2G8, clone 4C5 (Abnova Corporation), clone 4B8 (antibodies-online), clone MIH26 (Thermo Scientific Pierce Antibodies), clone UMAB61 (OriGene Technologies), clone 330104 (R&D Systems), clone 1B4 (Lifespan Biosciences), clone 440205, clone 5E7 (Creative Diagnostics) or from any one of the anti-BTLA antibodies described in WO2016/176583 (including clone 6F4), WO2011/014438 (including 8D5, 8A3, 20H4, 21H6, 15C5, 19A7 and 4C7), WO2010/106051 (including CNCM deposit number 1-4123) and WO2008/076560 (including 1B4, E4H9, 3C2, 3C2a, 6A5, 11E2, E8D9, 10H6 and 4C9 as detailed in Example 2); the sequences and features of the anti-BTLA antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds hVEM, e.g. human hVEM. In one embodiment, the HVEM antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from any one of the anti-HVEM antibodies described in WO2008/083169 (including LBH1); the sequences and features of the anti-BTLA antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CSF1R. In one embodiment, the CSF1R antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from any one of the anti-CSF1R antibodies described in WO2009/026303 (including 1.2, 1.109, 2.360 and 1.2.SM and the antibodies in FIGS. 1 and 2 ), WO2009/112245 (including CXIIG6), WO2011/070024 (including Mab 2F11, 2E10, 2H7 and 1G10, and their derivatives), WO2011/107553 (including 7H5.2G10/DSM ACC2922), WO2011/123381 (including antibody 1 and antibody 2), WO2011/131407 (including 7G5.3B6/DSM ACC2921), WO2011/140249 (including 0301, 0302, and 0311 their derivatives and the antibodies in tables 2, 3 and 5), WO2013/169264 & WO2014/036357 & WO2016/106180 & WO2016/168149 (including huAb1 to huAb16), WO2012/110360 & WO2013/057281 (including CXIIG6, H19K12, H27K5 and H27K15 and the humanised antibodies of tables 1 and 2), WO2013/087699 (including 9D11.2E8 and 10H2.2F12), WO2014/072441 (including H27K15), WO2014/173814 & WO2013/132044 (including Mab 2F11, Mab 2E10, Mab 2H7, Mab 1G10 and sc2-4A5 and the antibodies in Table 3 and 3b), WO2015/028455 & WO2015/028454 (including Ab535, Ab969, and derivatives, e.g. Ab969.g2), WO2015/036511 & WO2016/207312 (including 2F11, 2E10 and the derivatives described in embodiment 33) and WO2017/049038 (including ALM-423 and the antibodies listed in Table 2); the sequences and features of the anti-CSF1R antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CD39. In one embodiment, the CD39 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from BY40, BY12, BA54g (Biolegend), BU61 (Santa Cruz Biotech), A1 (Ebiosciences), AC2 (Immunotech), 22A9 (Abcam), 24DMS1 or any one of the anti-CD39 antibodies described in WO96/32471, WO00/04041, WO01/10205 (including CD39L4), WO2009/09547 (including CNCM-I-3889/BY40), WO2014/169255, WO2012/085132 (including antibodies VY12, BY40 and BA54g), WO2016/073845 (including R29-5-13A, R29-5-71A, R29-5-165C and R29-9-8B), WO2017/089334 (including 1-391, 1-392 and antibodies produced from hybridomas 1-3889 and CNCM I-41171) and WO2009/095478; the sequences and features of the anti-CD39 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CD40, e.g. human CD40. In one embodiment, the CD40 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from BMS3h-56-269, CP-870,893, dacetuzumab, SEA-CD40, ADC-1013, R07009789 and Chi Lob 7/4, or from any one of the anti-CD40 antibodies described in WO2017/059243, WO2017/059196, WO2017/040932, WO2017/040566, WO2017/004016, WO2017/004006, WO2016/196314, WO2016/028810, WO2016/023960, WO2016/023875, WO2015/134988, WO2015/091853, WO2014/070934, WO2014/065403, WO2014/065402, WO2014/04298, WO2013/164789, WO2013/034904, WO2012/149356, WO2012/145673, WO2012/125569, WO2012/111762, WO2012/075111, WO2012/065950, WO2012/041635, WO2011/123489, WO2010/123012, WO2010/104761, WO2010/121231, WO2009/062125, WO2010/104747, WO2010/104748, WO2010/104749, WO2010/024676, WO2009/094391, WO2009/062054, WO2008/091954, WO2007/130493, WO2007/129895, WO2007/124299, WO2007/053767, WO2007/053661, WO2006/128103, WO2006/073443, WO2005/063981, WO2005/063289 (US2012/0263732), WO2005/044855, WO2005/044306, WO2005/044294, WO2005/044307, WO2005/044304, WO2005/044854, WO2005/044305, WO03/040170 (U.S. Pat. No. 7,563,442B, 7,618,633B, 7,338,660B, 7,288,251B, 7,626,012B, 8,388,971B, US2013/0024956), WO03/029296, WO02/088186, WO01/83755, WO02/28905, WO02/28480, WO02/28481, WO02/28904, WO01/37870, WO01/16180, WO00/75348 and WO99/42075, WO97/31025, WO95/17202 and WO95/09653; the sequences and features of the anti-CD40 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CD73. In one embodiment, the CD73 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the VH, or the VL or the VH and VL region from 1E9 (Santa Cruz Biotechnology), AD2, 7G2, 4G4 or from any one of the anti-CD73 antibodies described in WO2017/064043 (including 7H10, 12F9, 15D7, 4611, 11D9 and 9D2), WO2016/081748 (including 4C3, 7A11, 6E11, 5F8, 4C3, 11F11, 11A6, CD73.4-1, CD73.4-2, CD73.3, 11F11-1, 11F11-2, 11F11, 4C3-1, 4C3-2, 4C3-3, 4D4, 10D2-1, 10D2-2, 11A6, 24H2, 5F8-1, 5F8-2 and 5F8-3), WO2016/131950 (including 11E1, 8C7, 3C12 and 6E1), WO2016/075176 (including MEDI9447, clone 10.3 and clone 2C5) & WO2016/075099 (including CD730004, CD730008, CD7300011, CD730021, CD730042, CD730046, CD730047, CD730068 and CD730069), WO2016/055609 (including 11E1, 6E1, 3C12 and 8C7); the sequences and features of the anti-CD73 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CD96. In one embodiment, the CD96 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region of 6A6, or NK92.39 (E bioscience), 1C8, 3H8, MAA6359 or from any one of the anti-CD96 antibodies described in WO2008/073316, WO2009/007124, WO2013/184912, WO2014/089169, WO2014/149310 (including antibody 3.3), WO2015/024060 or WO2015/024042, WO2015/024060 (including mAb 3.3); the sequences and features of the anti-CD96 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CXCR2. In one embodiment, the CXCR2 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from any one of the anti-CXCR2 antibodies described in WO2015/169811 (including HY29 and HY29GL), WO2014/170317 (including CX2-Mab #1 to #19), WO2012/062713, WO2013/168108 (including 163D2-127D1, 163E3-127D1, 163E3-54B12, 163D2-54B12, 2B2-163E3, 2B2-163D2, 97A9-2B2, 97A9-54B12, 127D1-163D2, 127D1-163E3, 2B2-97A9, 54B12-163D2, 54B12-163E3, 163D2-2B2, 163E3-2B2, 127D1-97A9, 54B12-97A9, 97A9-127D1 and derivatives thereof), WO2009/117706 (including 48311.211, 5E8/CXCR2, clone 19 and derivatives thereof), WO2009/120186 (including RII115, 48311 and derivatives thereof) and WO2002/26249; the sequences and features of the anti-CXCR2 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CD200. In one embodiment, the CD200 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region DX-109, samalizumab/ALXN-6000, TTI-200.7 or from any one of the anti-CD200 antibodies described in WO99/24565 (including M3B5 and the antibodies in Examples 4 and 5), WO02/11762 (including 3B6 and the antibodies in the Examples), WO2004/060295 (US2004/0213783), WO2004/078938 (including scFv-9), WO2006/020266 (U.S. Pat. No. 8,840,885B2, including CG1R3A10, cG2aR3A10, cG2aR3B7, dGIR3A5, dGIR3B5, and dGIR3B10 and the antibodies described in FIGS. 9A-9C, FIGS. 21A and 21B), WO2007/084321 (U.S. Pat. No. 8,709,415B2, including ALXN5200, hB7VH3VL2, C2aB7G1, C2aB7G2/G4, V3V2-G1 and V3V2-G2/G4), WO2009/014745 (including OX90mG2a (FIG. 10 ), OX90NE and OX90NE-AG), and WO2011/100538 & US2013/0189258 (including Antibody 1 and Antibody 2); the sequences and features of the anti-CD200 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CCR4, e.g. human CCR4. In one embodiment, the CCR4 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from mogamulizumab, KM3060 (see Niwa et al., 2004, Cancer Research 64, 2127-2133), and KW-0761 (see Ishida et al., Annals of Oncology 2008, vol 19, supplement 4, 513) or from any one of the anti-CCR4 antibodies described in WO2016/178779 & WO2016/057488 (including mAb2-3, 1-44, 1-49, 2-1 and 2-2), WO2015/179236 (including KW-0761), WO2013/166500 (including mAb1567, c1567, h1567, mAb 1-4 and 2-3 and the antibodies in Examples 6 and 13), WO2012/076883 (including antibodies 208, 306, 308, 406, 501, 503, 601, 603 and 803—Tables 1-9), WO2010/142952 (including 17G, 9E, 11F, 9E10, 9E10J and 9E1D—see Tables 1-16), WO2009/086514 (including mAb1567 and the humanised mAbs in Example 14), WO2005/035582 (including the DG44/CCR4 antibody and the Ms705/CCR4 antibody (FERM BP-8467)), WO2005/053741 & WO01/64754 (US6,989,145B, US7,666,418B, US8,197,814B, US8,632,996B, including KM2160 (FERM BP-10090), KM2760 (FERM deposit BP-7054)), WO2003/018635 (including KM2160, KM8759 (FERM BP-8129) and KM8760 (FERM BP-8130), WO00/42074 (US6,488,930B, US7,138,117B, including 2610, 10E4, 1G1 and the antibodies deposited as ATCC accession number HB-12624 and HB-12625) and WO00/41724 (US6,881,406B, US6,245,332B, including 1G1 and the antibody deposited under ATCC accession number HB-12624); the sequences and features of the anti-CCR4 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CXCL9, e.g. human CXCL9. In one embodiment, the CXCL9 antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from mAb 392-100 or AF392 (R&D Systems).

In one embodiment, the antigen-binding site specifically binds CXCL10. In one embodiment, the CXCL10 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region of mAb266 (R & D systems) or from any one of the anti-CXCL10 antibodies described in WO017/8708 (including CR.G (IP-10) (IgG1) (PharMingen) ande IP-10 (IgG)(A.Luster), WO02/15932, WO03/006045, WO2004/082714, WO2004/045525, WO2004/045526, WO2004/101511 (including antibodies in table 1 and AIP12, HuAIP12, MuAIP12, AIP13, HuAIP13, MuAIP13, AIP6, AIP8, AIP14, AIP18, AIP21, AIP22, ATPS and AIP17), WO2005/060457 (including AIP5, AIP6, AIP8, AIP10, AIP12, AIP13, AIP14, AIP17, AIP18, AIP21, AIP22, AIP32 and AIP36), WO2005/011605, WO2005/023201, WO2005/058815 (including 1D4, 1E1, 2G1, 3C4, 6A5, 6A8, 6B10, 7C10, 8F6, 10A12 and 10A12S13C4), WO2005/084708, WO2006/039819, WO2006/118085, WO2008/047486, WO2008/044824 (including antibodies #124, #31, #28, #43 and #137), WO2008/106200, WO2009/023566, WO2012/149320 (including MSX-1100 and 6A5), WO2014/003742 (including the antibody of Example 14), WO2013/170735, WO2014/189306, WO2015/063187; the sequences and features of the anti-CXCL10 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CD155, e.g. human CD155. In one embodiment, the CD155 antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from clone SKII.4 (BioLegend).

In one embodiment, the antigen-binding site specifically binds an immune activator. In one embodiment, the antigen-binding site specifically binds an immune activator selected from CD137, GITR, OX40, CD40, CXCR3 (e.g. agonistic activity against CXCR3), CD3 and ICOS (e.g. agonistic activity against ICOS). In one embodiment, the antigen-binding site specifically binds an immune activator selected from ICOS, CD137, GITR and OX40.

In one embodiment, the antigen-binding site specifically binds CD137, e.g. hCD137. In one embodiment, the CD137 antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from urelumab, BMS-663513, PF-05082566 (Pfizer), 1D8 and 3E1, 4B4 (BioLegend 309809), H4-1BB-M127 (BD Pharmingen 552532), BBK.2 (Thermo Fisher M S621PABX), 145501 (Leinco Technologies B591), the antibody produced by cell line deposited as ATCC No. HB-11248 (U.S. Pat. No. 6,974,863) or XmAb-5592, or from any one of the anti-CD137 antibodies described in WO2017/04945, WO2016/134358, WO2015/179236, WO2012/177788, WO2012/145183, WO2012/032433, WO2009/135019, WO2005/035584, U.S. Pat. No. 6,974,863, WO2004/055513 and WO2004/010947; the sequences and features of the anti-CD137 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds GITR, e.g. hGITR. In one embodiment, the GITR antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from MK4166, TRX518, TRX385, MAB689 (R & D Systems), YGITR765 (Novus Biologicals) or 1D8 (Novus Biologicals), or from any one of the anti-GITR antibodies described in WO2015/187835 (including 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and 6G10), WO2015/184099 (including 1042-7, 32-15, 1039-45, 1333-21, 231-1039-45, 231-32-15, Hum231 #1, Hum231 #2, m6C8, pab1964, to pab1973, pab1975 to pab1977, pab1979 to pab1981, pab1983, pab2159, pab2160, pab2161 and the antibodies in tables 1 and 2), WO2015/031667 (including antibodies Ab1 to Ab59 in table 1), WO2015/026684 (including an antibody with a CDR sequence of Seq ID 1-66), WO2013/039954 (including, 2155, 1718, 1649, 1362, 954, 827, 698, 706 and antibodies listed in Tables 1 & 3), WO2011/051726 (including antibodies containing CDRs a-f listed on page 17), WO2011/028683 (including antibodies 36E5, 61F6, 61G6, 3D6, 6H6, 1D8, 17F10, 35D8, 49A1, 9E5, 31H6 and antibodies from hybridomas PTA-9889, PTA-9890, PTA-9891, PTA-9892, PTA-9893, PTA-10286, PTA-10287, PTA-10288, PTA-10289, PTA-10290, and PTA-10291), WO2009/009116 (including antibody 2F8), WO2007/133822 (including antibodies listed in Table 1), WO2006/105021 (including 6C8, 2F8, HuN6C8-Agly, HuQ6C8-Gly, and HuQ6C8-Agly), WO2006/050172 & WO2004/084942 (including DTA-1), WO03/006058 (including anti-GITR/TNFRSF18 #AF524), WO2016/054638 (including mAb #1-81, #3-167, #5-139, #7-192, #10-116, #11-126, #12-46, #13-169, #14-182, #15-68 and #17-60), WO2016/196792 (including 6G10, 28F3, 19D3, 18E10, 3C3, 2G6, 8A6, 9G7, 14E3 and 19H8), WO2017/087678 (including 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2 and 6G10); the sequences and features of the anti-GITR antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds OX40, e.g. hOX40. In one embodiment, the OX40 antigen-binding site comprises the CDRH1, CDRH2, CDR3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from GSK3174998, L106 BD (Pharmingen Product #340420), ACT35 (Santa Cruz Biotechnology, Catalog #20073), MOXR0916, MEDI-6469, MEDI-0562, 9612 (Weinberg, A. D., et al., J Immunother 29, 575-585 (2006)), the humanised anti-OX40 Ab described in Morris et al., Mol Immunol. May 2007; 44(12):3112-3121, or from any one of the anti-OX40 antibodies described in WO2017/077085 (including SAP9, SAP28.2, SAP15.3, SAP29-50, SAP25-29 and SAP29-23 and humanised versions described in Examples 4 and 5), WO2017/063162 (including O3, O19, O21 and the affinity matured version in Example 5—Table 2, including 21 #H28H33, 21 #H65, 21 #H96, 21 #VHnew-L80, 21 #H96-L80), WO2017/050729 (including SP197), WO2017/021912 & WO2017/021910 (including ANTIBODY 106-222, OX86, and the antibodies described in FIGS. 6 and 7 ), WO2016/200836 & WO2016/200835 (including MOXR0916/1A7.gr1 IgG1), WO2016/196228 (including 3F4, 14B6-1, 14B6-2, 23H3, 18E9, 8B11, 20B3, 20C1, 6E1-1, 6E1-2, 14A2, 14A2-1, 14A2-2, L106, OX40.1, OX40.5, OX40.8, OX40.6, and OX40.16 and OX40.21—FIGS. 1 to 10 ), WO2016/179517 (including 11D4, pab1949, pab1949-1, pab2044, pab2193-1, Tables 1 to 4), WO2016/057667 (including 9612 and OX40mAb24), WO2015/153513 (including 3C8, 1D2, 1A7 and their variants described in the sequence listing, including A1A7.gr1 and 3C8.gr.5, the antibodies described in FIG. 1 ), WO2014/148895 (including ACT35, 12H3, 12H3 (FIG. 25 )—and humanised versions VL1H1, VL1VH2, VL1VH3, VL2H1, VL2VH2 and VL2VH3 (FIGS. 43 & 44 ) and 20E5 (FIG. 24 )), WO2013/068563 (including A26 [FIG. 2 ]), WO2013/038191 (including ACT35, 12H3 and 12H3), WO2013/028231 (including 119-122, 119-43-1, 106-222 and the antibodies in Table 1), WO2013/008171 (including 2F8, 1D4 and their derivatives, including VH6A/L9, and the antibodies in FIGS. 4 and 5 and tables 6 and 7), WO2012/027328 (including 119-122, 119-43-1, Hu106 and Hu106-222), WO2010/096418 (including A26), WO2008/106116 (including the antibodies in Tables 1 and 2, and A10 (inc A10A-F), B66—FIG. 14 —B2, B24, B36, B37, and B39) and WO2007/062245 (including 112V8 (ATCC No. PTA-7219), 112Y55 (ATCC No. PTA-7220), 112Y131 (ATCC No. PTA-7218), 112F32 (ATCC No. PTA-7217) and 112Z5 (ATCC No. PTA-7216); the sequences and features of the anti-OX40 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CXCR3, e.g. CXCR3. In one embodiment, the CXCR3 antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from GSK3174998 or from any one of the anti-CXCR3 antibodies described in WO2016/200836, WO2016/200835, WO2016/196228, WO2016/179517, WO2016/057667, WO2015/153513, WO2014/148895, WO2013/068563, WO2013/038191, WO2013/028231, WO2013/008171, WO2012/027328, WO2010/096418, WO2011/073180, WO2008/106116 and WO2007/062245; the sequences and features of the anti-CXCR3 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CD27, e.g. hCD27. In one embodiment, the CD27 antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from any one of the anti-CD27 antibodies described in WO2016/145085 (including 1F5), WO2015/016718 (including hCD27.15 and 1F5), WO2014/140374 (including 2F2, 5F24, 5F32, 10F13, 10F31, 11F26, 1052 to 015, F2A4B2 and their derivatives, including hz5F24VH+V5Q, hz5F24VL+K45Q), WO2013/138586 (including C2177, C2186, C2191, and C2192 and the derivatives in Examples 8 to 12, and tables 7 to 42), WO2012/004367 (including hCD27.15/ATCC number PTA-11008), WO2011/130434 (including 1G5, 1H8, 3H12, 3H8, 2G9, 1F5, 3A10, 2C2, ms 1A4, ms 9F4 and ms M-T271), WO2011/081164 & WO2010/001908 (including KM4027, KM4028, KM4026, KM4030, KM4032 and derivatives thereof), WO2008/051424 (including LG3A10 and AT124-1); the sequences and features of the anti-CD27 antibodies are incorporated herein by reference.

In one embodiment, the antigen-binding site specifically binds CD3, e.g. hCD3. In one embodiment, the CD3 antigen-binding site comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3, or the V_(H), or the V_(L) or the V_(H) and V_(L) region from OKT3 antibody, otelixizumab, teplizumab or visilizumab, or from any one of the anti-CD3 antibodies described in WO2017/010874, WO2017/009442, WO2016/204966, WO2016/180721, WO2016/179003, WO2016/116626, WO2016/014974, WO2015/104346, WO2015/095392, WO2015/001085, WO2014/047231, WO2013/188693, WO2013/186613, WO2013/158856, WO2012/173819, WO2012/162067, WO2005/118635, WO2004/108158, WO2004/052397, WO2004/024771, WO01/51644, WO00/05268, WO97/44362, WO93/19196, WO92/06193 and WO91/09968; the sequences and features of the anti-CD3 antibodies are incorporated herein by reference.

By providing one molecule having binding sites for (at least) two antigens—ICOS and PD-L1—the present invention facilitates administration of multi-agent therapies to patients. Thus, in situations where it would be impractical and/or unfeasibly expensive to treat a patient with a cocktail of different therapeutic agents, a patient may instead by treated using the multispecific antibody, optionally in combination with one or more other therapies, thereby reducing the number of different drug compositions that are administered to the patient. As noted, the other therapy may comprise administration of an antibody to another target antigen, such as those antigens and antibodies listed above.

The term “in combination” in the context of the administration of other therapies refers to the use of more than one therapy. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject with a disease. A first therapy can be administered before (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) the administration of a second therapy to a subject. Any additional therapy can be administered in any order with the other additional therapies. In certain embodiments, the antibodies of the invention can be administered in combination with one or more therapies (e.g., therapies that are not the antibodies of the invention that are currently administered to prevent, treat, manage, and/or ameliorate a disease. Non-limiting examples of therapies that can be administered in combination with an antibody of the invention include analgesic agents, anaesthetic agents, antibiotics, or immunomodulatory agents or any other agent listed in the U.S. Pharmacopoeia and/or Physician's Desk Reference.

Treatment with an immunomodulatory antibody such as anti-CTLA4, anti-PD1 or anti-PDL1, especially one with Fc effector function (e.g., a human IgG1), may create an environment in which further depletion of ICOS highly expressing immune-suppressive cells is beneficial. It may be advantageous to combine the multispecific antibody of the invention with such an immunomodulator to enhance its therapeutic effects.

A patient who has been treated with an immunomodulatory antibody (e.g., anti-PDL-1, anti-PD-1, anti-CTLA-4) may particularly benefit from treatment with the multispecific antibody. One reason for this is that an immunomodulatory antibody may increase the number of ICOS-positive Tregs (e.g., intratumoural Tregs) in the patient. This effect is also observed with certain other therapeutic agents, such as recombinant IL-2. Anti-ICOS treatment may reduce and/or reverse a surge or rise in ICOS+ Tregs (e.g., intratumoural Tregs) resulting from treatment of the patient with another therapeutic agent. A patient selected for treatment with the multispecific antibody may thus be one who has already received treatment with a first therapeutic agent, the first therapeutic agent being an antibody (e.g., immunomodulator antibody) or other agent (e.g., IL-2) that increases the number of ICOS+ Tregs in the patient.

The multispecific antibody may be used in combination with an antagonist of PD-1 or an antibody (e.g., antagonist antibody) to PD-1. The anti-PD-1 antibody may be any disclosed herein, e.g., pembrolizumab or nivolumab. The anti-PD-1 antibody may be one that inhibits binding of PD-1 to PD-L1 and/or PD-L1.

The multispecific antibody may be used in combination with an antagonist of CTLA-4 or an antibody (e.g., antagonist antibody) to CTLA-4. The anti-CTLA-4 antibody may be any disclosed herein, e.g., ipilimumab or tremelimumab. The anti-CTLA-4 antibody may be one that inhibits binding of CTLA-4 to CD80 and/or CD86.

Antibodies to PD-L1

The antigen-binding site of any anti-PD-L1 antibody may be used in a multispecific antibody. A multispecific antibody may be used in combination with an anti-PD-L1 antibody, or used to treat a patient who has previously received treatment with an anti-PD-L1 antibody. Numerous examples of anti-PD-L1 antibodies are disclosed herein and others are known in the art. Characterisation data for many of the anti-PD-L1 antibodies mentioned here has been published in U.S. Pat. Nos. 9,567,399 and 9,617,338, both incorporated by reference herein.

1D05 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:34. 1D05 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:44. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No: 526, Seq ID No:528, Seq ID No: 530, Seq ID No: 532 or Seq ID No: 534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36). A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

84G09 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:13, comprising the CDRH1 amino acid sequence of Seq ID No:7 (IMGT) or Seq ID No:10 (Kabat), the CDRH2 amino acid sequence of Seq ID No:8 (IMGT) or Seq ID No:11 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:9 (IMGT) or Seq ID No:12 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:14. 84G09 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:23, comprising the CDRL1 amino acid sequence of Seq ID No:17 (IMGT) or Seq ID No:20 (Kabat), the CDRL2 amino acid sequence of Seq ID No:18 (IMGT) or Seq ID No:21 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:19 (IMGT) or Seq ID No:22 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:24. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:15 (heavy chain nucleic acid sequence Seq ID No:16). A full length light chain amino acid sequence is Seq ID No:25 (light chain nucleic acid sequence Seq ID No:26).

1D05 HC mutant 1 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:47, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 1 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:44. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

1D05 HC mutant 2 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:48, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 2 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:44. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

1D05 HC mutant 3 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:49, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 3 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:44. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

1D05 HC mutant 4 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:342, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 4 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:44. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

1D05 LC mutant 1 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:34. 1D05 LC mutant 1 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:50, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The CDRL2 sequence of 1D05 LC Mutant 1 is as defined by the Kabat or IMGT systems from the V_(L) sequence of Seq ID No:50. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205 or Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36).

1D05 LC mutant 2 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:34. 1D05 LC mutant 2 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:51, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36).

1D05 LC mutant 3 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:34. 1D05 LC mutant 3 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:298, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The CDRL2 sequence of 1D05 LC Mutant 3 is as defined by the Kabat or IMGT systems from the V_(L) sequence of Seq ID No:298. The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:44. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205 or Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36). A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

411B08 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:58, comprising the CDRH1 amino acid sequence of Seq ID No:52 (IMGT) or Seq ID No:55 (Kabat), the CDRH2 amino acid sequence of Seq ID No:53 (IMGT) or Seq ID No:56 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:54 (IMGT) or Seq ID No:57 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:59. 411B08 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:68, comprising the CDRL1 amino acid sequence of Seq ID No:62 (IMGT) or Seq ID No:65 (Kabat), the CDRL2 amino acid sequence of Seq ID No:63 (IMGT) or Seq ID No:66 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:64 (IMGT) or Seq ID No:67 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:69. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:60 (heavy chain nucleic acid sequence Seq ID No:61). A full length light chain amino acid sequence is Seq ID No:70 (light chain nucleic acid sequence Seq ID No:71).

411C04 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:78, comprising the CDRH1 amino acid sequence of Seq ID No:72 (IMGT) or Seq ID No:75 (Kabat), the CDRH2 amino acid sequence of Seq ID No:73 (IMGT) or Seq ID No:76 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:74 (IMGT) or Seq ID No:77 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:79. 411C04 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:88, comprising the CDRL1 amino acid sequence of Seq ID No:82 (IMGT) or Seq ID No:85 (Kabat), the CDRL2 amino acid sequence of Seq ID No:83 (IMGT) or Seq ID No:86 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:84 (IMGT) or Seq ID No:87 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:89. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:80 (heavy chain nucleic acid sequence Seq ID No:81). A full length light chain amino acid sequence is Seq ID No:90 (light chain nucleic acid sequence Seq ID No:91).

411D07 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:98, comprising the CDRH1 amino acid sequence of Seq ID No:92 (IMGT) or Seq ID No:95 (Kabat), the CDRH2 amino acid sequence of Seq ID No:93 (IMGT) or Seq ID No:96 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:94 (IMGT) or Seq ID No:97 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:99. 411D07 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:108, comprising the CDRL1 amino acid sequence of Seq ID No:102 (IMGT) or Seq ID No:105 (Kabat), the CDRL2 amino acid sequence of Seq ID No:103 (IMGT) or Seq ID No:106 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:104 (IMGT) or Seq ID No:107 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:109. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:100 (heavy chain nucleic acid sequence Seq ID No:101). A full length light chain amino acid sequence is Seq ID No: 110 (light chain nucleic acid sequence Seq ID No:111).

385F01 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:118, comprising the CDRH1 amino acid sequence of Seq ID No:112 (IMGT) or Seq ID No:115 (Kabat), the CDRH2 amino acid sequence of Seq ID No:113 (IMGT) or Seq ID No:116 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:114 (IMGT) or Seq ID No:117 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:119. 385F01 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:128, comprising the CDRL1 amino acid sequence of Seq ID No:122 (IMGT) or Seq ID No:125 (Kabat), the CDRL2 amino acid sequence of Seq ID No:123 (IMGT) or Seq ID No:126 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:124 (IMGT) or Seq ID No:127 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:129. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:120 (heavy chain nucleic acid sequence Seq ID No:121). A full length light chain amino acid sequence is Seq ID No:130 (light chain nucleic acid sequence Seq ID No:131).

386H03 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:158, comprising the CDRH1 amino acid sequence of Seq ID No:152 (IMGT) or Seq ID No:155 (Kabat), the CDRH2 amino acid sequence of Seq ID No:153 (IMGT) or Seq ID No:156 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:154 (IMGT) or Seq ID No:157 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:159. 386H03 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:168, comprising the CDRL1 amino acid sequence of Seq ID No:162 (IMGT) or Seq ID No:165 (Kabat), the CDRL2 amino acid sequence of Seq ID No:163 (IMGT) or Seq ID No:166 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:164 (IMGT) or Seq ID No:167 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:169. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:160 (heavy chain nucleic acid sequence Seq ID No:161). A full length light chain amino acid sequence is Seq ID No:170 (light chain nucleic acid sequence Seq ID No:171).

389A03 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:178, comprising the CDRH1 amino acid sequence of Seq ID No:172 (IMGT) or Seq ID No:175 (Kabat), the CDRH2 amino acid sequence of Seq ID No:173 (IMGT) or Seq ID No:176 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:174 (IMGT) or Seq ID No:177 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:179. 389A03 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:188, comprising the CDRL1 amino acid sequence of Seq ID No:182 (IMGT) or Seq ID No:185 (Kabat), the CDRL2 amino acid sequence of Seq ID No:183 (IMGT) or Seq ID No:186 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:184 (IMGT) or Seq ID No:187 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:189. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:180 (heavy chain nucleic acid sequence Seq ID No:181). A full length light chain amino acid sequence is Seq ID No:190 (light chain nucleic acid sequence Seq ID No:191).

413D08 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:138, comprising the CDRH1 amino acid sequence of Seq ID No:132 (IMGT) or Seq ID No:135 (Kabat), the CDRH2 amino acid sequence of Seq ID No:133 (IMGT) or Seq ID No:136 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:134 (IMGT) or Seq ID No:137 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:139. 413D08 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:148, comprising the CDRL1 amino acid sequence of Seq ID No:142 (IMGT) or Seq ID No:145 (Kabat), the CDRL2 amino acid sequence of Seq ID No:143 (IMGT) or Seq ID No:146 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:144 (IMGT) or Seq ID No:147 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:149. The VH domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No: 140 (heavy chain nucleic acid sequence Seq ID No:141). A full length light chain amino acid sequence is Seq ID No:150 (light chain nucleic acid sequence Seq ID No:151).

413G05 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:244, comprising the CDRH1 amino acid sequence of Seq ID No:238 (IMGT) or Seq ID No:241 (Kabat), the CDRH2 amino acid sequence of Seq ID No:239 (IMGT) or Seq ID No:242 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:240 (IMGT) or Seq ID No:243 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:245. 413G05 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:254, comprising the CDRL1 amino acid sequence of Seq ID No:248 (IMGT) or Seq ID No:251 (Kabat), the CDRL2 amino acid sequence of Seq ID No:249 (IMGT) or Seq ID No:252 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:250 (IMGT) or Seq ID No:253 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:255. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:246 (heavy chain nucleic acid sequence Seq ID No:247). A full length light chain amino acid sequence is Seq ID No:256 (light chain nucleic acid sequence Seq ID No:257).

413F09 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:264, comprising the CDRH1 amino acid sequence of Seq ID No:258 (IMGT) or Seq ID No:261 (Kabat), the CDRH2 amino acid sequence of Seq ID No:259 (IMGT) or Seq ID No:262 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:260 (IMGT) or Seq ID No:263 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:265. 413F09 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:274, comprising the CDRL1 amino acid sequence of Seq ID No:268 (IMGT) or Seq ID No:271 (Kabat), the CDRL2 amino acid sequence of Seq ID No:269 (IMGT) or Seq ID No:272 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:270 (IMGT) or Seq ID No:273 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:275. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:266 (heavy chain nucleic acid sequence Seq ID No:267). A full length light chain amino acid sequence is Seq ID No:276 (light chain nucleic acid sequence Seq ID No:277).

414B06 has a heavy chain variable (V_(H)) region amino acid sequence of Seq ID No:284, comprising the CDRH1 amino acid sequence of Seq ID No:278 (IMGT) or Seq ID No:281 (Kabat), the CDRH2 amino acid sequence of Seq ID No:279 (IMGT) or Seq ID No:282 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:280 (IMGT) or Seq ID No:283 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:285. 414B06 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:294, comprising the CDRL1 amino acid sequence of Seq ID No:288 (IMGT) or Seq ID No:291 (Kabat), the CDRL2 amino acid sequence of Seq ID No:289 (IMGT) or Seq ID No:292 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:290 (IMGT) or Seq ID No:293 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:295. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:286 (heavy chain nucleic acid sequence Seq ID No:287). A full length light chain amino acid sequence is Seq ID No:296 (light chain nucleic acid sequence Seq ID No:297).

416E01 has a heavy chain variable region (V_(H)) amino acid sequence of Seq ID No:349, comprising the CDRH1 amino acid sequence of Seq ID No:343 (IMGT) or Seq ID No:346 (Kabat), the CDRH2 amino acid sequence of Seq ID No:344 (IMGT) or Seq ID No:347 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:345 (IMGT) or Seq ID No:348 (Kabat). The heavy chain nucleic acid sequence of the V_(H) domain is Seq ID No:350. 416E01 has a light chain variable region (V_(L)) amino acid sequence of Seq ID No:359, comprising the CDRL1 amino acid sequence of Seq ID No:353 (IMGT) or Seq ID No:356 (Kabat), the CDRL2 amino acid sequence of Seq ID No:354 (IMGT) or Seq ID No:357 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:355 (IMGT) or Seq ID No:358 (Kabat). The light chain nucleic acid sequence of the V_(L) domain is Seq ID No:360. The V_(H) domain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The V_(L) domain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:351 (heavy chain nucleic acid sequence Seq ID No:352). A full length light chain amino acid sequence is Seq ID No:361 (light chain nucleic acid sequence Seq ID No:362).

Treatment of Diseases and Conditions

Antibodies according to the present invention may be used in methods of treatment of the human body by therapy. The treatment may be treatment of cancer or a number of other conditions exemplified herein.

The antibodies find use in increasing effector T cell response, which is of benefit for a range of diseases or conditions, including treating cancers or solid tumours and in the context of vaccination. Increased Teff response may be achieved using an antibody that modulates the balance or ratio between Teffs and Tregs in favour of Teff activity.

The antibodies may be used for depleting regulatory T cells and/or increasing effector T cell response in a patient, and may be administered to a patient to treat a disease or condition amenable to therapy by depleting regulatory T cells and/or increasing effector T cell response.

An antibody of the present invention, or a composition comprising such an antibody molecule or its encoding nucleic acid, may be used or provided for use in any such method. Use of the antibody, or of a composition comprising it or its encoding nucleic acid, for the manufacture of a medicament for use in any such method is also envisaged. The method typically comprises administering the antibody or composition to a mammal. Suitable formulations and methods of administration are described elsewhere herein.

One envisaged therapeutic use of the antibodies is treatment of cancer. The cancer may be a solid tumour, e.g., renal cell cancer (optionally renal cell carcinoma, e.g., clear cell renal cell carcinoma), head and neck cancer, melanoma (optionally malignant melanoma), non-small cell lung cancer (e.g., adenocarcinoma), bladder cancer, ovarian cancer, cervical cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, testicular germ cell carcinoma, or the metastases of a solid tumour such as those listed, or it may be a liquid haematological tumour e.g., lymphoma (such as Hodgkin's lymphoma or Non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma, DLBCL) or leukaemia (e.g., acute myeloid leukaemia). An anti-ICOS antibody may enhance tumour clearance in melanoma, head and neck cancer and non-small cell lung cancer and other cancers with a moderate to high mutational load [32]. By enhancing patients' immune response to their neoplastic lesions, immunotherapy using an anti-ICOS antibody offers the prospect of durable cures or long-term remissions, potentially even in the context of late stage disease.

Cancers are a diverse group of diseases, but anti-ICOS antibodies offer the possibility of treating a range of different cancers by exploiting the patient's own immune system, which has the potential to kill any cancer cell through recognition of mutant or overexpressed epitopes that distinguish cancer cells from normal tissue. By modulating the Teff/Treg balance, anti-ICOS antibodies can enable and/or promote immune recognition and killing of cancer cells. While anti-ICOS antibodies are therefore useful therapeutic agents for a wide variety of cancers, there are particular categories of cancers for which anti-ICOS therapy is especially suited and/or where anti-ICOS therapy can be effective when other therapeutic agents are not.

One such group is cancer that is positive for expression of ICOS ligand. Cancer cells may acquire expression of ICOS ligand, as has been described for melanoma [33]. Expression of ICOS ligand may provide the cells with a selective advantage as the surface-expressed ligand binds ICOS on Tregs, promoting the expansion and activation of the Tregs and thereby suppressing the immune response against the cancer. Cancer cells expressing ICOS ligand may depend for their survival on this suppression of the immune system by Tregs, and would thus be vulnerable to treatment with anti-ICOS antibodies that target the Tregs. This applies also to cancers derived from cells that naturally express ICOS ligand. Continued expression of ICOS ligand by these cells again provides a survival advantage through immune suppression. A cancer expressing ICOS ligand may be derived from antigen-presenting cells such as B cells, dendritic cells and monocytes and may be a liquid haematological tumour such as those mentioned herein. Interestingly it has been shown that these types of cancer are also high in ICOS and FOXP3 expression (TCGA data).

Accordingly, the multispecific antibodies can be used in methods of treating cancers that are positive for expression of ICOS ligand. Further, a cancer to be treated with anti-ICOS antibody according to the present invention may be one that is positive for expression of ICOS and/or FOXP3, and optionally also expresses ICOS ligand.

Patients may undergo testing to determine whether their cancer is positive for expression of the protein of interest (e.g., ICOS ligand, ICOS and/or FOXP3), for example by taking a test sample (e.g., tumour biopsy) from the patient and determining expression of the protein of interest. Patients whose cancer has been characterised as positive for expression of one, two or all such proteins of interest are selected for treatment with anti-ICOS antibody. As discussed elsewhere herein, anti-ICOS antibody may be used as a monotherapy or in combination with one or more other therapeutic agents.

Anti-ICOS antibodies also offer hope to patients whose cancers are refractory to treatment with antibodies or other drugs directed to immune checkpoint molecules such as CTLA-4, PD-1, PD-L1, CD137, GITR or CD73. These immunotherapies are effective against some cancers but in some cases a cancer may not respond, or it may become unresponsive to continued treatment with the antibody. In common with antibodies to immune checkpoint inhibitors, anti-ICOS antibodies modulate the patient's immune system—nevertheless an anti-ICOS antibody may succeed where such other antibodies fail. It is shown herein that animals carrying A20 B cell lymphomas could be treated with anti-ICOS antibodies to reduce growth of the tumour, shrink the tumour and indeed clear the tumour from the body, whereas treatment with an anti-PD-L1 antibody was no better than control. The A20 cell line has also been reported to be resistant to anti-CTLA-4 [34].

Accordingly, the antibodies of the present invention can be used in methods of treating cancers that are refractory to treatment with one or more immunotherapies, such as (any or all of) an anti-CTLA-4 antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody, anti-GITR antibody, or anti-CD73 antibody. A cancer may be characterised as being refractory to treatment with an antibody or other drug if treatment with that antibody or drug does not significantly reduce growth of the cancer, e.g., if a tumour continues to grow or does not reduce in size or if after a response period the tumour re-initiates its growth. Non-response to a therapeutic agent may be determined ex vivo by testing a sample (e.g., tumour biopsy sample) for cancer cell killing or growth inhibition, and/or in the clinical setting by observing (e.g., using an imaging technology, including MRI) that a patient treated with the therapy is not responding to treatment. Patients whose cancer has been characterised as refractory to treatment with such an immunotherapy are selected for treatment with anti-ICOS antibody.

Further, the antibodies may be used to treat B-cell derived cancer that is resistant to treatment with an anti-CD20 antibody. Anti-ICOS antibodies represent a treatment for cancers that fail to respond to, or become resistant to, therapy with anti-CD20 antibodies like rituximab. Anti-ICOS antibody may be used as a second-line (or further, or additional) treatment for such cancers. The anti-CD20 antibody resistant cancer may be a B cell cancer, e.g., B cell lymphoma, such as diffuse large B cell lymphoma. Resistance of a cancer to anti-CD20 may be determined ex vivo by testing a sample (e.g., tumour biopsy sample) for cancer cell killing or growth inhibition by anti-CD20 antibody, and/or in the clinical setting by observing that a patient treated with the anti-CD20 antibody is not responding to treatment. Alternatively, or additionally, the cancer (e.g., a tumour biopsy sample) may be tested to assess expression of CD20, where an absence or low level of CD20 expression indicates loss of sensitivity to anti-CD20 antibody.

Samples obtained from patients may thus be tested to determine surface expression of a protein of interest, for example ICOS ligand, ICOS, FOXP3 and/or a target receptor to which another therapeutic agent (e.g., anti-receptor antibody) is directed. The target receptor may be CD20 (to which anti-CD20 antibody therapy such as rituximab is directed), or another receptor such as PD1, EGFR, HER2 or HER3. Surface expression of ICOS ligand, ICOS, FOXP3 and/or lack or loss of surface expression of the target receptor is an indication that the cancer is susceptible to anti-ICOS antibody therapy. Anti-ICOS antibodies can be provided for administration to a patient whose cancer is characterised by surface expression of ICOS ligand, ICOS, FOXP3 and/or lack or loss of surface expression of a target receptor, optionally where the patient has been previously treated with anti-CTLA4, anti-PD1, anti-PD-L1 or with an antibody to the target receptor and has not responded or has stopped responding to treatment with that antibody, as measured for example by continued or renewed cancer cell growth, e.g., increase in tumour size.

Any suitable method may be employed to determine whether cancer cells test positive for surface expression of a protein such as ICOS ligand, CD20 or other target receptors mentioned herein. A typical method is immunohistochemistry, where a sample of the cells (e.g., a tumour biopsy sample) is contacted with an antibody for the protein of interest, and binding of antibody is detected using a labelled reagent—typically a second antibody that recognises the Fc region of the first antibody and carries a detectable label such as a fluorescent marker. A sample may be declared to test positive where at least 5% of cells are labelled, as visualised by cell staining or other detection of the label. Optionally a higher cut-off such as 10% or 25% may be used. The antibody will generally be used in excess. Reagent antibodies to the molecules of interest are available or may be generated by straightforward methods. To test for ICOS ligand, the antibody MAB1651 is currently available from R&D systems as a mouse IgG that recognises human ICOS ligand. To test for CD20 expression, rituximab may be used. Detection of mRNA levels of the ICOS ligand or target receptor of interest is an alternative technique [33].

A further indication that a tumour will respond to treatment with antibody according to the invention is the presence of Tregs in the tumour microenvironment. Activated Tregs are characterised by ICOS-high and Foxp3-high surface expression. The presence of Tregs in a tumour, especially in elevated numbers, provides a further basis on which a patient may be selected for treatment with the multispecific antibody. Tregs may be detected in a tumour biopsy sample ex vivo, for example by immunohistochemistry (assaying for co-expression of both Foxp3 and ICOS, using antibodies to the target protein followed by detection of labels, as described above) or by single cell dispersion of the sample for use in FACS with labelled antibodies to ICOS and Foxp3. FACS methods are exemplified in Example 17 and Example 18.

Antibodies according to the present invention may be used for treating cancers associated with infectious agents, such as virally-induced cancers. In this category are head and neck squamous cell carcinoma, cervical cancer, Merkel cell carcinoma and many others. Viruses associated with cancer include HBV, HCV, HPV (cervical cancer, oropharyngeal cancer), and EBV (Burkitts lymphomas, gastric cancer, Hodgkin's lymphoma, other EBV positive B cell lymphomas, nasopharyngeal carcinoma and post transplant lymphoproliferative disease). The International Agency for Research on Cancer (Monograph 100B) identified the following major cancer sites associated with infectious agents:

-   -   Stomach/Gastric: Helicobacter pylori     -   Liver: Hepatitis B virus, hepatitis C virus (HCV), Opisthorchis         viverrini, Clonorchis sinensis     -   Cervix uteri: Human papillomavirus (HPV) with or without HIV     -   Anogenital (penile, vulva, vagina, anus): HPV with or without         HIV     -   Nasopharynx: Epstein-Barr virus (EBV)     -   Oropharynx: HPV with or without tobacco or alcohol consumption     -   Kaposi's sarcoma: Human herpes virus type 8 with or without HIV     -   Non-Hodgkin lymphoma: H. pylori, EBV with or without HIV, HCV,         human T-cell lymphotropic virus type 1     -   Hodgkin's lymphoma: EBV with or without HIV     -   Bladder: Schistosoma haematobium.

Antibodies according to the present invention may be used for treating cancer associated with or induced by any of these infectious agents, such as the cancers specified above.

Stimulation of effector T cell response can also contribute to immunity against infectious disease and/or to recovery from infectious disease in a patient. Thus, an anti-ICOS antibody may be used for treating infectious disease by administering the antibody to a patient.

Infectious diseases include those caused by pathogens, e.g., bacterial, fungal, viral or protozoal pathogens, and treatment may be to promote immune response in a patient against the pathogen infection. An example of a bacterial pathogen is tuberculosis. Examples of viral pathogens are hepatitis B and HIV. Examples of protozoal pathogens are Plasmodium species, which cause malaria, such as P. falciparum.

The antibody may be used for treating infections, e.g., infection by any pathogen mentioned herein. Infection may be persistent or chronic infection. Infection may be localised or systemic. Extended contact between a pathogen and the immune system may lead to exhaustion of the immune system or development of tolerance (manifested for example through increased levels of Tregs, and tipping of the Treg:Teff balance in favour of Tregs) and/or to immune evasion by the pathogen, through evolution and modification of displayed pathogen antigens. These features reflect similar processes that are believed to occur in cancer. Anti-ICOS antibodies present a therapeutic approach to treating infection by a pathogen, e.g., chronic infection, through modulation of the Treg:Teff ratio in favour of Teff and/or other effects described herein.

Treatment may be of patients who have been diagnosed as having an infectious disease or an infection. Alternatively, treatment may be preventative, and administered to a patient to guard against contracting a disease, e.g., as a vaccine, as described elsewhere herein.

Further exemplary cancers in humans include a Merkel cell carcinoma, breast cancer, prostate cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer (e.g. glioblastoma), cervical cancer, choriocarcinoma, colon and rectum cancer, connective tissue cancer, cancer of the digestive system; endometrial cancer, esophageal cancer; eye cancer; cancer of the head and neck; nasopharyngeal cancer; gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g. small cell and non-small cell); lymphoma including Hodgkin's and Non-Hodgkin's lymphoma including but not limited to DLBCL; Chronic lymphocytic leukaemia, melanoma; uveal melanoma, myeloma, neuroblastoma, oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer, retinoblastoma; rhabdomyosarcoma; rectal cancer, renal cancer (renal cell carcinoma (RCC)), cancer of the respiratory system; sarcoma, skin cancer; stomach cancer, testicular cancer, thyroid cancer; uterine cancer, cancer of the urinary system, as well as other carcinomas and sarcomas. Further examples of virally induced cancers including; Nasopharyngeal carcinoma, certain Types of NHL (for example but not limited to EBV+ CNS lymphomas, DLBCL and BL, Hodgkins lymphoma (thought to be EBV driven) HPV-related cervical and head an neck squamous cell carcinomas); HBV hepatocellular carcinoma.

Exemplary chronic infections in humans include HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV).

Proliferative or invasion-related diseases that can be treated with the antibodies or antigen binding fragments described herein include neoplastic diseases, and the metastasis associated with such neoplastic disease, such as, melanoma, uveal melanoma, skin cancer, small cell lung cancer, non-small cell lung cancer, salivary gland, glioma, hepatocellular (liver) carcinoma, gallbladder cancer, thyroid tumour, bone cancer, gastric (stomach) cancer, prostate cancer, breast cancer (including triple negative breast cancer), ovarian cancer, cervical cancer, uterine cancer, vulval cancer, endometrial cancer, testicular cancer, bladder cancer, lung cancer, glioblastoma, thyroid cancer, endometrial cancer, kidney cancer, colon cancer, colorectal cancer, pancreatic cancer, esophageal carcinoma, brain/CNS cancers, neuronal cancers, head and neck cancers (including but not limited to squamous cell carcinoma of the head and neck (SCCHN)), mesothelioma, sarcomas, biliary (cholangiocarcinoma), small bowel adenocarcinoma, pediatric malignancies, epidermoid carcinoma, sarcomas, cancer of the pleural/peritoneal membranes and leukaemia, including acute myeloid leukaemia, acute lymphoblastic leukaemia, and multiple myeloma. Treatable chronic viral infections include HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) in humans, simian immunodeficiency virus (Sly) in monkeys, and lymphocytic choriomeningitis virus (LCMV) in mice.

The can be administered alone, or in combination with other antibodies or chemo therapeutic drugs, radiation therapy or therapeutic vaccines. In one embodiment, the antibody or antigen binding fragment thereof is administered as an antibody-drug conjugate in which the antibody or antigen binding fragment thereof is linked to a drug moiety such as a cytotoxic or cytostatic agent. The use of antibody-drug conjugates for the local delivery of cytotoxic or cytostatic agents in the treatment of cancer allows targeted delivery of the drug moiety to tumours, and intracellular accumulation therein, where systemic administration of unconjugated drug may result in unacceptable levels of toxicity. Drugs in antibody drug conjugates can include, but are not limited to, daunomycin, doxorubicin, methotrexate, and vindesine. Toxins can also be used in antibody-toxin conjugates, including, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin. The toxins may effect their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase.

Still further embodiments include methods of treating a proliferative or invasion-related disease in a mammal by administering to the animal a therapeutically effective dose of an antibody or antigen binding fragment thereof. In another embodiment, the antibodies or antigen binding fragments thereof can be used in a method for treating a mammal suffering from a disease selected from: neoplastic or non-neoplastic disease, chronic viral infection, and a malignant tumour, wherein the method includes administering to the mammal a therapeutically effective dose of an antibody or antigen binding fragment thereof.

Still further embodiments include methods of treating a disease of immunological dysfunction in a mammal by administering to the animal a therapeutically effective dose of an antibody or antigen binding fragment thereof as described herein. Exemplary immunological dysfunction in humans includes diseases of neurological deficit, such as Alzheimer's disease.

It has further been proposed that an immune response, particularly an IFNγ-dependent systemic immune response, could be beneficial for treatment of Alzheimer's disease and other CNS pathologies that share a neuroinflammatory component. WO2015/136541 proposes treatment of Alzheimer's disease using an anti-PD-1 antibody (also see Baruch K. et al., PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of Alzheimer's disease, Nature Medicine, 2016, 22(2):137-137).

A multispecific antibody, or composition comprising one, may be used in treating or preventing a disease or condition (which may be a hPD-L1-mediated disease or condition), e.g. selected from neoplastic or non-neoplastic disease, chronic viral infections, and malignant tumours, such as melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell cancer, bladder cancer, head and neck squamous cell carcinoma, mesothelioma, virally induced cancers (such as cervical cancer and nasopharyngeal cancer), soft tissue sarcomas, haematological malignancies such as Hodgkin's and non-Hodgkin's disease and diffuse large B-cell lymphoma (for example melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell cancer, bladder cancer, head and neck squamous cell carcinoma and mesothelioma or for example virally induced cancers (such as cervical cancer and nasopharyngeal cancer) and soft tissue sarcomas). The antibody may be used for the manufacture of a medicament for treating any such condition or others mentioned herein. Methods of treating such a condition in a human are also described, including methods comprising administering to said human a therapeutically effective amount of the antibody, wherein the disease or condition is thereby treated or prevented.

A hPD-L1 mediated disease or condition may be cancer or any disease or condition described herein. In one embodiment, the hPD-L1 mediated disease is a virally induced cancer, such as cervical cancer and nasopharyngeal cancer, for example cervical cancers caused by HPV infection. In one embodiment, the hPD-L1 mediated disease is a chronic viral infection. In one embodiment, the hPD-L1 mediated disease is a neoplastic disease. In one embodiment, the hPD-L1 mediated disease is a non-neoplastic disease. In one embodiment, the hPD-L1 mediated disease is a malignant tumour. In one embodiment, the hPD-L1 mediated disease is a cancer which is known to be responsive to PD-L1 therapy, such as melanoma, Merkel cell carcinoma, non-small cell lung cancer (squamous and non-squamous), renal cell cancer, bladder cancer, head and neck squamous cell carcinoma, mesothelioma. In one embodiment, the hPD-L1 mediated disease is a cancer which is a soft tissue sarcoma.

The hPD-L1-mediated disease or condition may alternatively be a neurodegenerative disease, disorder or condition, optionally wherein the neurodegenerative disease, disorder or condition is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, corticobasal degeneration, Rett syndrome, a retinal degeneration disorder selected from age-related macular degeneration and retinitis pigmentosa; anterior ischemic optic neuropathy, glaucoma, uveitis, depression, trauma-associated stress or post-traumatic stress disorder, frontotemporal dementia, Lewy body dementias, mild cognitive impairments, posterior cortical atrophy, primary progressive aphasia and progressive supranuclear palsy or aged-related dementia, in particular Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease, and e.g. Alzheimer's disease.

Methods of treatment may further comprise administering to the human a further therapy, for example a further therapeutic agent, optionally wherein the further therapeutic agent is independently selected from the group consisting of:

-   -   a. other immune checkpoint inhibitors (such as anti-TIM-3         antibodies, anti-CTLA-4 antibodies, anti-TIGIT antibodies and         anti-LAG-3 antibodies);     -   b. immune stimulators (such as anti-OX40 antibodies, anti-GITR         antibodies, anti-CD137 antibodies, anti-ICOS antibodies and         anti-CD40 antibodies);     -   c. chemokine receptor antagonists (such as CXCR4, CCR4 and         CXCR2);     -   d. targeted kinase inhibitors (such as CSF-1R or VEGFR         inhibitors);     -   e. angiogenesis inhibitors (such as anti-VEGF-A or Delta-like         Ligand-4);     -   f. immune stimulating peptides or chemokines (such as CXCL9 or         CXCL10);     -   g. cytokines (such as IL-15 and IL-21);     -   h. bispecific T-cell engagers (BiTEs) having at least one         specificity against CD3 (e.g. CD3/CD19 BiTE);     -   i. other bi-specific molecules (for example IL-15-containing         molecules targeted towards tumour associated antigens, for         example Epidermal growth factor receptors such as EGFR, Her-2,         New York Esophageal Cancer-1 (NY-ESO-1), GD2, EpCAM or Melanoma         Associated Antigen-3 (MAGE-A3));     -   j. oncolytic viruses (such as HSV virus (optionally which         secretes GMCSF), Newcastle disease virus and Vaccinia virus);     -   k. vaccination with tumour associated antigens (such as New York         Esophageal Cancer-1 [NY-ESO-1], Melanoma Associated Antigen-3         [MAGE-3]);     -   l. cell-based therapies (such as chimeric Antigen Receptor-T         cells (CAR-T) for example expressing anti-CD19, anti-EpCam or         anti-mesothelin);     -   m. bispecific NK cell engagers having a specificity against an         activating MK receptor such as NKG2D or CD16a; and     -   n. adoptive transfer of tumour specific T-cells or LAK cells,         -   or optionally wherein the further therapy is chemotherapy,             radiotherapy and surgical removal of tumours. Radiotherapy             may be single dose or in fractionated doses, either             delivered to affected tissues directly or to the whole body.

Chemotherapeutic agents may any as described hereinabove, in particular agents that induce immunogenic cell death, for example platinum therapies, such as oxaliplatin. In one embodiment, the chemotherapy is a standard of care cytotoxic chemotherapy for the cancer being treated.

The further therapeutic agents of this concept may be delivered by any method, which methods are well-known to those skilled in the art. For example, the further therapeutic agents may be delivered orally, systemically or locally (to the tumour environment). In one embodiment, the further therapeutic agent is delivered orally. In one embodiment, the further therapeutic agent is delivered systemically (e.g. intravenously). In one embodiment, the further therapeutic agent is delivered locally to the tumour environment.

Compositions and routes of administration are described in more detail hereinbelow.

The further therapeutic agent is administered sequentially or simultaneously with the multispecific antibody.

A pharmaceutical composition may comprise a multispecific antibody as described herein and a pharmaceutically acceptable excipient, diluent or carrier, optionally further comprising a further therapeutic agent independently selected from the group consisting of:

-   -   a) other immune checkpoint inhibitors (such as anti-TIM-3         antibodies, anti-CTLA-4 antibodies, anti-TIGIT antibodies and         anti-LAG-3 antibodies);     -   b) immune stimulators (such as anti-OX40 antibodies, anti-GITR         antibodies, anti-CD137 antibodies, anti-ICOS antibodies and         anti-CD40 antibodies);     -   c) chemokine receptor antagonists (such as CXCR4, CCR4 and         CXCR2);     -   d) targeted kinase inhibitors (such as CSF-1R or VEGFR         inhibitors);     -   e) angiogenesis inhibitors (such as anti-VEGF-A or Delta-like         Ligand-4);     -   f) immune stimulating peptides or chemokines (such as CXCL9 or         CXCL10);     -   g) cytokines (such as IL-15 and IL-21);     -   h) bispecific T-cell engagers (BiTEs) having at least one         specificity against CD3 (e.g. CD3/CD19 BiTE);     -   i) other bi-specific molecules (for example IL-15-containing         molecules targeted towards tumour associated antigens, for         example Epidermal growth factor receptors such as EGFR, Her-2,         New York Esophageal Cancer-1 (NY-ESO-1), GD2, EpCAM or Melanoma         Associated Antigen-3 (MAGE-A3));     -   j) oncolytic viruses (such as HSV virus (optionally which         secretes GMCSF), Newcastle disease virus and Vaccinia virus);     -   k) vaccination with tumour associated antigens (such as New York         Esophageal Cancer-1 [NY-ESO-1], Melanoma Associated Antigen-3         [MAGE-3]);     -   l) cell-based therapies (such as chimeric Antigen Receptor-T         cells (CAR-T) for example expressing anti-CD19, anti-EpCam or         anti-mesothelin);     -   m) bispecific NK cell engagers having a specificity against an         activating MK receptor such as NKG2D or CD16a;     -   and     -   n) adoptive transfer of tumour specific T-cells or LAK cells.

Pharmaceutical formulations are well-known to those skilled in the art. In one embodiment, the antibody or fragment is administered intravenously. In one embodiment, the antibody or fragment is administered subcutaneously.

In an example, an antibody or fragment as disclosed herein is contained in a medical container, e.g., a vial, syringe, IV container or an injection device (such as an intraocular or intravitreal injection device). In an example, the antibody or fragment is in vitro, for example, in a sterile container.

In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Such compositions, however, may be administered by a route other than intravenous. Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The further therapeutic agents of this concept may be delivered by any method, which methods are well-known to those skilled in the art. For example, the further therapeutic agents may be delivered orally, systemically or locally (to the tumour environment). In one embodiment, the further therapeutic agent is delivered orally. In one embodiment, the further therapeutic agent is delivered systemically (e.g. intravenously). In one embodiment, the further therapeutic agent is delivered locally to the tumour environment.

Encoding Nucleic Acids and Methods of Expression

Isolated nucleic acid may be provided, encoding antibodies according to the present invention. Nucleic acid may be DNA and/or RNA. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode an antibody.

The present invention provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. Exemplary nucleotide sequences are included in the sequence listing. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

The present invention also provides a recombinant host cell that comprises one or more nucleic acids encoding the antibody. Methods of producing the encoded antibody may comprise expression from the nucleic acid, e.g., by culturing recombinant host cells containing the nucleic acid. The antibody may thus be obtained, and may be isolated and/or purified using any suitable technique, then used as appropriate. A method of production may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals.

The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is E. coli. Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others.

Vectors may contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Nucleic acid encoding an antibody can be introduced into a host cell. Nucleic acid can be introduced to eukaryotic cells by various methods, including calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. Introducing nucleic acid in the host cell, in particular a eukaryotic cell may use a viral or a plasmid based system. The plasmid system may be maintained episomally or may be incorporated into the host cell or into an artificial chromosome. Incorporation may be either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by expressing the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene, then optionally isolating or purifying the antibody.

Nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques.

The present invention also provides a method that comprises using nucleic acid described herein in an expression system in order to express an antibody.

EXAMPLES

A number of bispecific antibodies were generated, using different antibody formats, and with different anti-ICOS and anti-PD-L1 binding sites. Bispecific antibodies were shown to bind and induce an agonistic signal at ICOS, to bind to PD-L1 inhibiting the interaction with PD-1, and to deplete cells expressing high levels of ICOS.

Anti-ICOS Fv regions of antibodies, and anti-PD-L1 Fv regions of antibodies, can be generated using the Kymouse—a transgenic mouse technology platform. Kymouse covers the entire human immunoglobulin (Ig) repertoire of V, D and J genes required to make fully human antibodies. On selection and recovery of recombined variable regions, the antibodies are re-formatted to yield antibodies with isotypes or Fc-domains of choice. STIM001 and STIM003 are anti-ICOS antibodies originating from the Kymouse. Their Fv regions were included in bispecific antibody formats as described in these Examples.

The example anti-PD-L1 binding domains that were used in the mAb² bispecific antibodies described in these Examples were produced as Fcabs, utilising permissive residues in the CH3 domain of the constant chain of human IgG1 termed the AB, CD and EF loops to generate IgG-based Fcabs which bind PD-L1.

Example 1 Generation of ICOS/PD-L1 Bispecific Antibody FIT-Ig

Bispecific antibodies for ICOS and PD-L1 were generated in FIT-Ig format, as illustrated in FIG. 2 .

The anti-PD-L1/ICOS tetravalent bispecific FIT-Ig molecule combines the variable regions of an anti-ICOS antibody with the variable regions of an anti-PD-L1 antibody. The bispecific molecule presents two Fabs in tandem, fused with an Fc. The molecules are symmetrical.

In this example, the anti-ICOS antibody domains were those of STIM003 and the anti-PD-L1 antibody domains were those of Antibody W. The FIT-Ig molecules were generated with anti-ICOS binding specificity as the “outer” Fab (antibody A in FIG. 2 ) or with anti-ICOS binding specificity as the “inner” Fab (antibody B in FIG. 2 ). Fc domains were either human IgG1 or mouse IgG2a. Thus a total of four different FIT-Ig molecules were produced using the STIM003 and AbW binding specificities:

-   -   ICOS/PD-L1 hIgG1     -   ICOS/PD-L1 mIgG2a     -   PD-L1/ICOS hIgG1     -   PD-L1/ICOS mIgG2a

For each FIT-Ig molecule, the three constructs shown in FIG. 2 (ii) were generated, and cloned into an expression vector (pTT5) via restriction enzymes and sequences were verified. A 30 mL HEK transient transfection was then performed, using the three constructs in 1:1:1 ratio. After 6 days, supernatant was analysed to assess the level of expression, quantifying using human IgG1 and mouse IgG2a as standards. Expression data are shown in Table E1-1 below.

TABLE E1-1 Expression of FIT-Ig in μg/mL. Standard human IgG1 STIM003_AbW_hIgG1 11.1 AbW_STIM003_hIgG1 52.5 Standard mouse IgG2a STIM003_AbW_mIgG2a 44.2 AbW_STIM003_mIgG2a 42.8 Sequences of further example FIT-Ig molecules are shown in Table S3. mAb²

Bispecific antibodies for ICOS and PD-L1 were generated in mAb² format, as illustrated in FIG. 3 . The following molecules were generated and expressed:

STIM001_289. A mAb² IgG1 in which the two Fab regions comprise the VH and VL domains of anti-ICOS antibody STIM001 and the Fcab region binds human PD-L1. STIM001_457. A mAb² IgG1 in which the two Fab regions comprise the VH and VL domains of anti-ICOS antibody STIM001 and the Fcab region binds mouse PD-L1. STIM003_289. A mAb² IgG1 in which the two Fab regions comprise the VH and VL domains of anti-ICOS antibody STIM003 and the Fcab region binds human PD-L1. STIM003_457. A mAb² IgG1 in which the two Fab regions comprise the VH and VL domains of anti-ICOS antibody STIM003 and the Fcab region binds mouse PD-L1. STIM003_574. A mAb² IgG1 in which the two Fab regions comprise the VH and VL domains of anti-ICOS antibody STIM003 and the Fcab region binds mouse PD-L1.

The Fcab regions of _457 and _574 have a minor variation in amino acid sequence. In vitro, the affinity of 457 to mouse PD-L1 is lower compared with that of 574. The affinity of 574 for mouse PD-L1 is closer to the affinity of 289 for human PD-L1. In vivo, 457 and 574 have similar anti-tumour efficacy.

The following control antibodies were also generated for use in the experiments:

lgG1_289. A mAb² IgG1 in which the two Fab regions do not bind ICOS or PD-L1, and the Fcab region binds human PD-L1. lgG1_457. A mAb² IgG1 in which the two Fab regions do not bind ICOS or PD-L1, and the Fcab region binds mouse PD-L1. lgG1_438. A mAb² IgG1 in which the two Fab regions do not bind ICOS or PD-L1, and the Fcab region binds mouse PD-L1. IgG1_438 and IgG1_457 have minor variation in the amino acid sequences of the antigen-binding loops in the Fcab and are functionally equivalent for the purposes of the assays described below.

The mAb² antibodies were generated as IgG1, containing IgG1 CH1, CH2 and CH3 constant regions, with the Fcab binding loops in the IgG1 CH3 constant region. Unless otherwise stated, IgG1 was wild type human IgG1. The “LAGA” variant IgG1 sequence was used where specified. The “LAGA” variant includes mutations L235A and G237A which disable Fc-mediated effects ADCC and CDC as described in WO99/58679 and in Shields et al. J. Biol. Chem., March 2; 276(9):6591-604 2001.

Example 2 Kinetic Surface Plasmon Resonance (SPR) Assay for Characterisation of Bispecific Antibody Binding to ICOS and PD-L1

Analysis of mAb² binding to human PD-L1 and mouse PD-L1 This kinetic SPR assay confirmed that addition of human variable regions to anti-PD-L1 Fcab molecules to create mAb² constructs did not affect the ability of the Fcab to recognise PD-L1. Six different mAb²_289 constructs exhibited similar binding to human PD-L1, and six different mAb²_457 constructs exhibited similar binding to mouse PD-L1.

Method:

An anti-human IgG capture surface was created on a Series S C1 chip (GE Healthcare, cat No BR100535). A cocktail of three anti-human IgG antibodies were covalently coupled to the biosensor chip surface (Jackson Labs: cat No 109-005-008; cat No 309-006-008 and cat No 109-006-008), using 10 mM Na Acetate pH 4.5 buffer as the diluent for the antibody.

For the kinetic assay, mAb² constructs were diluted to 5 μg/mL in running buffer (1×HBS-EP+ Buffer Technova, cat. No. H8022) and captured on the anti-human capture surface. The human recombinant extra cellular domain PD-L1 protein was used as analyte at 81 nM, 27 nM, 9 nM, 3 nM, 1 nM and 0 nM, and injected over the mAb²_289 constructs. The mouse recombinant extra cellular domain PD-L1 protein was injected as analyte at 243 nM, 81 nM, 27 nM, 9 nM, 3 nM, 1 nM and 0 nM over the mAb²_457 constructs. Finally, the surface was regenerated between each mAb² construct using 100 mM PO₄. The assay was carried out at 25° C.

The buffer injection (i.e. 0 nM) was used to double reference the sensorgrams. The analysis was carried out using the 1:1 binding model inherent to the Biacore 8K's analysis software.

Results:

Data for binding to human PD-L1 are shown in Table E2-1 below.

TABLE E2-1 mAb²_289 affinities for binding to human PD-L1. Five concentrations of human PD-L1 (81, 27, 9, 3 and 1 nM) were used as analyte over each mAb²_289 construct, captured at 5 μg/mL. ka (1/Ms) kd (1/s) KD (nM) hybrid control_289 8.99E+06 1.84E−03 0.20 hybrid control_289_LAGA 8.14E+06 1.40E−03 0.17 STIM003_289 5.31E+06 1.38E−03 0.26 STIM003_289_LAGA 8.69E+06 1.63E−03 0.19 STIM001_289 8.94E+06 1.87E−03 0.21 STIM001_289_LAGA 7.68E+06 1.47E−03 0.19

With respect to the K_(D) values, the binding of the mAb²_289 constructs to human PD-L1 was comparable and within the variance expected with this type of assay.

Data for binding to mouse PD-L1 are shown in Table E2-2 below.

TABLE E2-2 mAb²_457 affinities for binding to mouse PD-L1. Six concentrations of mouse PD-L1 (243, 81, 27, 9, 3 and 1 nM) were used as analyte over each mAb²_457 construct, captured at 5 μg/mL. ka (1/Ms) kd (1/s) KD (nM) STIM003_457 1.31E+06 1.07E−01 82.0 STIM003_457_LAGA 1.36E+06 1.30E−01 95.9 STIM001_457 1.32E+06 1.29E−01 97.8 STIM001_457_LAGA 2.11E+06 2.27E−01 107.8 hybrid control_457 1.35E+06 1.34E−01 99.3 hybrid control_457_LAGA 1.32E+06 1.43E−01 108.7

With respect to the K_(D) values, the binding of the mAb²_457 constructs to mouse PD-L1 is comparable. However, given that the apparent affinity of the interaction is in the 80-110 nM range and the top concentration of mouse recombinant extra cellular domain PD-L1 was only 243 nM, it is unlikely that a true saturated Rmax was achieved in this assay, hence the actual affinity may be lower than indicated by these data. Nevertheless it can be concluded that the constructs are comparable in their binding and the anti-PD-L1 Fcab retains its binding for mouse PD-L1 when incorporated into a mAb² format.

Avidity Surface Plasmon Resonance Analysis of mAb² Binding to Human ICOS

This avidity SPR assay confirmed that the bispecific mAb² constructs were able to bind ICOS. Values obtained for binding were similar across all samples tested and within experimental variance for this type of assay, where capture level, concentration and biophysical form can have an impact on values.

Method:

Biotinylated human ICOS protein diluted at 7.5 μg/mL in running buffer (1×HBS-EP+ Buffer Technova, cat. No. H8022) was captured on a NLC sensor chip (Bio-Rad, cat No 1765021). The surface was then blocked using biocytin (Sigma Aldrich, cat No B1758) at 1 mg/mL.

The ICOS/PD-L1 bispecific mAb² and the corresponding human anti-ICOS IgG1 constructs were injected as analyte at 500, 167, 56, 18.5 and 0 nM for the STIM001_mAb² and STIM001, and at 40, 10, 2.5, 0.625 and 0 nM for the STIM003_mAb² and STIM003. The assay was carried out at 25° C.

The buffer injection (i.e. 0 nM) was used to double reference the sensorgrams. The analysis was carried out using the equilibrium model inherent to the ProteOn's analysis software for each mAb² construct.

Results:

Data for STIM003 mAb² constructs binding to human ICOS are shown in Table E2-3.

TABLE E2-3 Apparent affinities of STIM003_mAb² for binding to human ICOS. Four concentrations of STIM003_mAb² and STIM003 (40, 10, 2.5 and 0.625 nM) were used as analyte over the human ICOS protein, captured at 7.5 μg/mL. Apparent Capture KD (nM) Calculated level from Off-rate On-rate mAb² (RU) equilibrium (1/s) (1/M · s) STIM003_289 528 0.7 2.65E−04 3.55E+05 STIM003_289_LAGA 548 0.6 2.78E−04 4.77E+05 STIM003_457 1122 0.9 2.44E−04 2.75E+05 STIM003_457_LAGA 935 0.8 2.32E−04 2.84E+05 STIM003 543 1.7 4.49E−04 2.67E+05

Data for STIM001 mAb² constructs binding to human ICOS are shown in Table E2-4.

TABLE E2-4 Apparent affinities of STIM001_mAb² for binding to human ICOS. Four concentrations of STIM001_mAb² and STIM001 (500, 167, 56 and 18.5 nM) were used as analyte over the human ICOS protein, captured at 7.5 μg/mL. Apparent Capture KD (nM) Calculated level from Off-rate On-rate mAb² (RU) equilibrium (1/s) (1/M · s) STIM001_289 712 19.1 4.18E−04 2.19E+04 STIM001_289_LAGA 1166 25.4 3.54E−04 1.39E+04 STIM001_457 1011 9.2 3.46E−04 3.76E+04 STIM001_457_LAGA 716 10.6 3.32E−04 3.13E+04 STIM001 730 57.1 4.56E−04 7.99E+03

In conclusion, these data indicate that the presence of the PD-L1 binding site in the bispecific molecule does not affect binding of the anti-ICOS Fab arms.

Example 3 ELISA Characterisation of Bispecific Antibody Binding to ICOS and PD-L1

Antibodies in mAb² format were assessed for binding to recombinant human ICOS, mouse ICOS, human PD-L1 and mouse PD-L1 proteins. Binding of the ICOS/PD-L1 bispecific mAb² antibodies to recombinant ICOS protein and recombinant PD-L1 protein was confirmed in this assay.

DELFIA ELISA Method:

Recombinant ICOS proteins, human ICOS-mFc or mouse ICOS-mFc (Chimerigen) were diluted in 1×PBS and added to Black Hi-bind plates (Griener) at 1 μg/ml, 50 μl/well. Recombinant PD-L1 proteins, human PD-L1-Flag-His or mouse-His were diluted in 1×PBS and added to Black Hi-bind plates (Griener) at 4 μg/ml, 50 μl/well. The plates were left overnight at 4° C. The next day plates were washed 3× with 300 μl/well of 1×PBS+0.1% Tween and blocked with 200 ul/well of 1×PBS+1% BSA blocking buffer for 1 hr at RT on a plate shaker. Plates were washed 3× with 300 μl/well of 1×PBS+0.1% Tween.

In general, antibodies in monoclonal and mAb² format were diluted in 1×PBS+0.1% BSA buffer and diluted 1 in 3 from starting working concentration of either 0.399 μM, 0.199 μM over a 11 point titration. However, human-PD-L1 mAb² antibodies were diluted in 1×PBS+0.1% BSA buffer and diluted 1 in 2 from a starting working concentration of 0.066 μM over an 11 point titration. Titrated antibodies were added to the plates, 50 μl/well and left to incubate for 1 hr at R.T on a plate shaker. Plates were washed 3× with 300 μl/well of 1×PBS+0.1% Tween.

DELFIA® Eu-labelled Anti-human IgG (Perkin-Elmer) was diluted 1:500 in DELFIA Assay buffer (Perkin-Elmer) and added to the assay plate (50 μl/well), left to incubate for 1 hr at RT on a plate shaker. Plates were then washed 3× with 300 μl/well 1×DELFIA wash buffer before the addition of 50 μl/well of DELFIA Enhancement Solution (Perkin-Elmer), incubated for a minimum of 5 minutes in the dark. After incubation, assay was read on Envision plate reader (Perkin Elmer), Time-resolved fluorescence (TRF) was measured at 615 nm.

Titration curves and EC50 values [M] were plotted using Graphpad (Prism). EC50 values were calculated by first transforming the data using equation X=Log(X). The transformed data was then fitted using nonlinear regression, using fitting algorithm, log (agonist) vs. response—variable slope (four parameters).

Results:

Data are summarised in Tables E3-1 to E3-4 and shown in FIG. 4 .

In the human ICOS ELISA assay STIM003_289 and STIM003_457 produced similar EC50 values to STIM003 (mean EC50 values; 0.63 nM±0.18 nM and 0.43±0.069 nM and 0.75±0.27 nM respectively).

In the human ICOS ELISA assay STIM001_289 and STIM001_457 produced similar EC50 values to STIM001 (mean EC50 values; 13.1 nM±6.5 nM and 12.5±3.12 nM and 28.9±11 nM respectively).

In the mouse ICOS ELISA assay STIM003_289 and STIM003_457 produced similar EC50 values to STIM003 (mean EC50 values; 0.42 nM±0.075 nM and 0.28±0.037 nM and 0.37±0.039 nM respectively).

STIM001_289 and STIM001_457 produced similar EC50 values in the human ICOS ELISA assay (mean EC50 values; 13.16 nM±6.50 nM and 12.55 nM±3.12 nM respectively).

STIM003_289 and STIM003_457 produced similar EC50 values in the mouse ICOS ELISA assay (mean EC50 values; 0.42 nM±0.075 nM and 0.28±0.037 nM respectively).

STIM003_289 and STIM003_457 gave more robust N=3 EC50 values due to a higher max assay signal and better sigmoidal curve, not observed for STIM001_289 and STIM001_457 in the mouse ICOS ELISA assay.

STIM001_289, STIM003_289 and hybrid control_289 produced similar EC50 values in the human PD-L1 ELISA assay (mean EC50 values; 1.57 nM±0.32 nM, 1.43 nM±0.16 nM and 1.45 nM±0.28 nM respectively).

STIM001_457, STIM003_457 produced similar EC50 values in the mouse PD-L1 ELISA assay (mean EC50 values; 3.84 nM±1.87 nM, 6.83 nM±1.38 nM respectively).

TABLE E3-1 hICOS N = 1 N = 2 N = 3 Best-Fit values EC50 (M) EC50 (M) EC50 (M) Mean (M) STDEV (M) SEM (M) STIM003_289 4.908E−10 5.622E−10 8.406E−10 6.312E−10 1.84804E−10 1.06697E−10 STIM001_289 9.812E−09 9.013E−09 2.066E−08 1.316E−08 6.50816E−09 3.75749E−09 STIM003_457 5.098E−10 3.800E−10 4.006E−10 4.301E−10 6.97426E−11 4.02659E−11 STIM001_457 1.315E−08 1.533E−08 9.169E−09 1.255E−08 3.12347E−09 1.80333E−09 STIM003 4.880E−10 1.037E−09 7.270E−10 7.507E−10 2.75385E−10 1.58994E−10 STIM001 2.002E−08 2.549E−08 4.128E−08 2.893E−08 1.10422E−08  6.3752E−09

TABLE E3-2 mICOS (N = 1) (N = 2) (N = 3) Best-Fit values EC50 (M) EC50 (M) EC50 (M) Mean (M) STDEV (M) SEM (M) STIM003_289 3.358E−10 4.775E−10  4.529E−10 4.221E−10 7.56844E−11 4.36964E−11 STIM001_289 2.057* 2.270E−09  2.163E−09 2.217E−09 7.55591E−11 4.36241E−11 STIM003_457 3.265E−10 2.518E−10  2.881E−10 2.888E−10 3.73468E−11 2.15622E−11 STIM001_457 2.360E−08  5.80E−07* 437.58* 2.360E−08 STIM003  4.16E−10  3.86E−10   3.38E−10 3.799E−10 3.94138E−11 2.27555E−11 STIM001  1.14E−08  1.87E−08   1.73E−09 1.140E−08  8.4999E−09 4.90742E−09 *data excluded due to incomplete curve fit.

TABLE E3-3 hPD-L1 N = 1 N = 2 N = 3 Best-Fit values EC50 (M) EC50 (M) EC50 (M) Mean (M) STDEV (M) SEM (M) STIM003_289 1.23E−09 1.53E−09 1.52E−09 1.43E−09 1.69084E−10 9.76206E−11 STIM001_289 1.48E−09 1.30E−09 1.93E−09 1.57E−09 3.25612E−10 1.87992E−10 Hybrid Control_289 1.16E−09 1.46E−09 1.73E−09 1.45E−09 2.89106E−10 1.66915E−10 AbV 8.34E−10 8.18E−10 1.13E−09 9.28E−10 1.76778E−10 1.02063E−10

TABLE E3-4 mPD-L1 N = 1 N = 2 N = 3 Best-values EC50 (M) EC50 (M) EC50 (M) Mean (M) STDEV (M) SEM (M) STIM003_457 5.81743E−09 8.40577E−09 6.28E−09 6.836E−09 1.380E−09 7.965E−10 STIM001_457  5.6504E−09 3.97431E−09 1.91E−09 3.845E−09 1.874E−09 1.082E−09 AbV    1.38E−09    9.99E−10 8.66E−10 1.080E−09 2.644E−10 1.527E−10

Example 4 FACS Characterisation of Bispecific Antibody Binding to Cells Expressing ICOS or PD-L1 CHO Human ICOS and CHO Mouse ICOS Binding Assay (Flow Cytometry)

Ability of the ICOS/PD-L1 mAb² to bind human ICOS and mouse ICOS on the surface of CHO cells was confirmed in this assay. STIM001_289 mAb² and STIM003_289 mAb² were assessed for binding to transfected human ICOS and mouse ICOS CHO cells. Antibody binding to cells was detected with anti-human IgG labelled AlexaFluor 647.

Method:

CHO-S cells transfected with either human ICOS or mouse ICOS were resuspended in FACS buffer (PBS+1% w/v BSA+0.1% w/v sodium azide) and transferred to a 96-well V-bottom plate (Greiner) at a density of 1×10⁵ cells per well. mAb and mAb² were titrated in FACS buffer, 1 in 3 dilution across 11 points from a starting working concentration of 400 nM. Plates were centrifuged at 300×g for 3 minutes, supernatant discarded and 50 μL mAb or mAb² solution were added to cells and incubated at 4° C. for 1 hour. Cells were washed with 150 μL of PBS and centrifuged at 300×g for 3 minutes, supernatant was discarded and cell pellet resuspended in 150 μL PBS added. This wash step was repeated twice. Bound mAb or mAb² was detected by addition of 50 μL of anti-human 647 (Jackson ImmunoResearch) diluted to 3 ug/ml in FACS buffer. Cells were incubated for 1 hour at 4° C. in the dark. Cells were washed with 150 μL of PBS and centrifuged at 300 g for 3 minutes, supernatant was discarded and cell pellet resuspended in 150 μL PBS added. This wash step was repeated twice. Cells were fixed with 25 μL 4% v/v paraformaldehyde, incubated for 20 minutes at 4° C., cells were pelleted by centrifugation at 300×g and the supernatant discarded. Cells were washed with 150 μL of PBS and centrifuged at 300 g for 3 minutes, supernatant was discarded and cell pellet resuspended in 150 μL PBS added. This wash step was repeated. Pelleted cells were resuspended in 110 μL 1×PBS. AlexaFluor 647 signal intensity (geometric mean) was measured by flow cytometry using a Beckman Coulter CytoFLEX instrument.

Results:

EC50 data for binding to human ICOS are shown in Table E4-1 below and in FIG. 5 (A).

TABLE E4-1 hICOS N = 1 N = 2 N = 3 Best-Fit value EC50 (M) EC50 (M) EC50 (M) Mean (M) STDEV (M) SEM (M) STIM003_289 5.696E−09 2.282E−09 6.792E−09 4.924E−09 2.352E−09 1.358E−09 STIM001_289 5.529E−09 2.982E−09 3.134E−09 3.882E−09 1.429E−09 8.249E−10 STIM003 7.220E−09 4.890E−09 4.485E−09 5.532E−09 1.476E−09 8.522E−10 STIM001 3.009E−08 1.380E−08 1.973E−08 2.121E−08 8.244E−09 4.760E−09

EC50 data for binding to mouse ICOS are shown in Table E4-2 below and in FIG. 5 (B).

TABLE E4-2 mICOS N = 1 N = 2 N = 3 Best-Fit values EC50 (M) EC50 (M) EC50 (M) Mean (M) STDEV (M) SEM (M) STIM003_457 8.923E−09 1.099E−08 1.187E−08 1.059E−08 1.514E−09 8.741E−10 STIM001_457 5.138E−09 3.530E−09 6.974E−09 5.214E−09 1.724E−09 9.951E−10 STIM003 6.803E−09 2.195E−09 7.275E−09 5.424E−09 2.807E−09 1.620E−09 STIM001 2.567E−08 5.063E−09 2.008E−08 1.694E−08 1.066E−08 6.152E−09

CHO Human PD-L1 Binding Assay (Flow Cytometry)

Ability of the ICOS/PD-L1 mAb² to bind human PD-L1 expressed on the surface of CHO cells was confirmed in this assay. Binding of the bispecific antibody to PD-L1 could be detected using labelled ICOS recombinant protein, confirming the ability of the bispecific antibodies to bind both PD-L1 and ICOS.

STIM001_289 and STIM003_289 and one isotype control (IgG1_289) were assessed for human PD-L1 binding using FACS. These were characterised using anti-human IgG and human ICOS labelled AlexaFluor 647 detection.

Method:

CHO-S cells untransfected (referred to as WT) or transfected with the cDNA coding for human PD-L1 were diluted in FACS buffer (PBS+1% w/v BSA+0.1% w/v sodium azide) and were distributed to a 96-well V-bottom plate (Greiner) at a density of 5×10⁴ cells per well. Antibody and mAb² titrations were prepared from 198 nM working concentration as a 1/3 dilution series in FACS buffer. Plates were centrifuged at 300×g for 3 minutes to supernatant aspirated. 50 μL antibody or mAb² solution were added to cells and incubated at 4° C. for 1 hour. Cells were washed with 150 μL of PBS and centrifuged at 300 g for 3 minutes. Supernatant was aspirated and 150 μL PBS added. This wash step was repeated. Presence of bound antibody or mAb² was detected by addition of 50 μL of Anti-Human PE (Jackson ImmunoResearch) diluted 1/500 in FACS buffer or human ICOS labelled AlexaFluor 647 diluted to 225 nM to each well. Cells were incubated for 1 hour at 4° C. in the dark. Cells were washed as previously described. To fix cells, 100 μL 4% v/v paraformaldehyde was added and cells incubated for 20 minutes at 4° C., cells were pelleted by centrifugation at 300×g and the plates resuspended in 100 μL FACS buffer. AlexaFluor 647 and PE (R-Phycoerythrin) signal intensity (geometric mean) was measured by flow cytometry using a Beckman Coulter CytoFLEX instrument.

Results:

Bispecific antibodies and isotype control produced similar EC50 values to each other in the anti-human IgG detection system (0.64 nM, 0.64 nM and 0.55 nM respectively)—FIG. 6 (A). Bispecific antibodies produced similar EC50 values to each other in the human ICOS labelled AlexaFluor 647 system (0.48 nM and 0.58 nM respectively)—FIG. 6 (B). As expected, the isotype control antibody did not bind ICOS.

Also as expected, monospecific antibodies STIM001, STIM003 and control IgG1 did not show binding to human PD-L1 with either anti-human IgG detection or human ICOS labelled AlexaFluor 647.

CHO Mouse PD-L1 Binding Assay (Flow Cytometry)

Ability of the ICOS/PD-L1 mAb² to bind mouse PD-L1 on the surface of CHO cells was confirmed in this assay. Binding of the bispecific antibody to PD-L1 could be detected using ICOS, confirming the ability of the bispecific antibodies to bind both PD-L1 and ICOS.

STIM001_457 and STIM003_457 and one isotype control (IgG1_438) were assessed for human PD-L1 binding using FACS. These were characterised using anti-human IgG and human ICOS labelled AlexaFluor 647 detection.

Method:

CHO-S cells untransfected (referred to as WT) or transfected with mPD-L1 were diluted in FACS buffer (PBS+1% w/v BSA+0.1% w/v sodium azide) and were distributed to a 96-well V-bottom plate (Greiner) at a density of 5×10⁴ cells per well. Monospecific mAb and bispecific mAb² titrations were prepared from 22 nM working concentration as a 1/3 dilution series in FACS buffer. Plates were centrifuged at 300×g for 3 minutes to supernatant aspirated. 50 μL antibody or mAb² solution were added to cells and incubated at 4° C. for 1 hour. Cells were washed with 150 μL of PBS and centrifuged at 300 g for 3 minutes. Supernatant was aspirated and 150 μL PBS added. This wash step was repeated. Presence of bound mAb or mAb² was detected by addition of 50 μL of Anti-Human IgG PE (Jackson ImmunoResearch) diluted 1/500 in FACS buffer or human ICOS labelled AlexaFluor 647 diluted to 25 nM to each well. Cells were incubated for 1 hour at 4° C. in the dark. Cells were washed as previously described. To fix cells, 100 μL 4% v/v paraformaldehyde was added and cells incubated for 20 minutes at 4° C., cells were pelleted by centrifugation at 300×g and the plates resuspended in 100 μL FACS buffer. AlexaFluor 647 and PE (R-Phycoerythrin) signal intensity (geometric mean) was measured by flow cytometry using a Beckman Coulter CytoFLEX instrument.

Results:

Bispecific antibodies and isotype control produced similar EC50 values to each other in the anti-human IgG detection system (0.89±0.64 nM, 0.47±0.23 nM and 0.59±0.20 nM respectively)—FIG. 7 (A)—and in the human ICOS labelled AlexaFluor 647 system (1.29±1.26 nM and 0.81±0.56 nM respectively)—FIG. 7 (B). The isotype control did show binding.

Monospecific antibodies STIM001, STIM003 and IgG1 did not show binding to human PD-L1 with either anti-human IgG detection or human ICOS labelled AlexaFluor 647.

Example 5a Biolayer Interferometry Determination of mAb2 Binding to Fcγ Receptors Materials:

Sensors coated with a commercial anti-human FAb-CH1 ligand (Pall ForteBio, cat No 18-5125) were hydrated for 10 min in running buffer (1×HBS-EP+ Buffer: Technova, cat. No. H8022).

The mAb²_289 constructs STIM001_289 and STIM003_289 were diluted to 45 μg/mL in running buffer.

The recombinant human FcγRI (1257-FC-050, R&D Systems), mouse FcγRI (2074-FC-050, R&D Systems) and mouse FcγRIV (1974-CD-050, R&D Systems) were diluted to 1 μM in running buffer. The recombinant human FcγRIIIa (4325-FC-050, R&D Systems) and mouse FcγRIII (1960-FC-050, R&D Systems) were diluted to 2 μM in running buffer. Finally, the recombinant human FcγRIIa (1330-CD-050, R&D Systems), human FcγRIIb/c (1875-CD-050, R&D Systems) and mouse FcγRIIb (1460-CD-050, R&D Systems) were diluted to 3 μM in running buffer.

The human recombinant extracellular domain PD-L1 protein was diluted to 1 μM in running buffer.

Method:

The anti-human FAb-CH1 sensors were regenerated with 100 mM PO₄ then equilibrated in running buffer. STIM001_289 and STIM003_289 were captured at 45 μg/mL on the sensors and then loaded with 1 μM of human PD-L1. Finally, the sensors were dipped into the Fcγ receptor solution. The sensors were then regenerated and equilibrated again, and the same protocol was repeated for each Fcγ receptor. The assay was carried out at 25° C.

Fc gamma Receptor STIM001_289 STIM003_289 Human FcγR I Binding Binding Human FcγR IIa Binding Binding Human FcγR IIb/c Binding Binding Human FcγR IIIa Binding Binding Mouse FcγR I Binding Binding Mouse FcγR IIb Binding Binding Mouse FcγR III Binding Binding Mouse FcγR IV Binding Binding In this assay, both mAb²_289 constructs (STIM001_289 and STIM003_289) demonstrated expected binding to all the individual Fcγ receptors.

Example 5b Engagement of Fc Receptor on ADCC Effector Cells by Fc Region of Bispecific Antibody to ICOS and PD-L1

This assay determines ability of the Fc region of mAb² to engage FcγRIIIa on effector cells.

Method:

Immediately prior to the assay CHO (target) cells expressing human ICOS or mouse ICOS were centrifuged and resuspended in RPMI 1640 (Promega)+4% Low IgG serum (Promega) and plated at 50000 cells/well (25 μl/well) in 96-well white bottom TC-treated plates (Costar).

All antibodies were serially diluted 1 in 3 over 9 points, in RPMI 1640+4% Low IgG serum. For assays with human ICOS expressing target cells, the antibodies were diluted from starting working concentration of 10 nM and for mouse ICOS expressing target cells the mAb² and mAb were diluted from a starting working concentration of 20 nM and 35 nM, respectively. Diluted antibodies (25 μl/well) were added to the target cells and left to incubate for 0.5 hrs at room temperature. Thawed Jurkat NFAT luciferase v-variant effector cells (Promega) were resuspended in RPMI 1640+4% low IgG serum and added to the target cell/antibody mixture at 10000 cells/well (25 μl/well). After overnight incubation at 37° C., 5% CO2, luciferase activity was measured by adding luminogenic BioGlo substrate at 75 μl/well (Promega) directly to the wells, plates incubated for 10 min in the dark and read on an Envision (Perkin Elmer) plate reader.

Relative light unit (RLU) values from the raw data (Envision reads) were first normalised to ‘fold of induction’ using the following equation.

${{Fold}{of}{induction}} = \frac{RLU({induced}) - RLU({background})}{RLU({background})}$

Mean and standard deviation ‘fold of induction’ values for each antibody concentration were plotted and curves were fitted using the GraphPad Prism 4-parameter log-logistic curve. The average ‘fold of induction’ values for each experiment were then used to plot the inter-experimental values (+/−SD) from all three experiments.

Results:

Data are summarised in Table E5-1 and in FIGS. 8A and B. STIM003_289 and STIM001_289 produced similar EC50 values in the human and mouse ICOS ADCC assay.

TABLE E5-1 hICOS N = 1 N = 2 N = 3 Best-Fit value EC50 (M) EC50 (M) EC50 (M) Mean (M) STDEV (M) SEM (M) STIM003_289 2.309E−10 4.009E−10 2.069E−10 2.796E−10 1.058E−10 6.108E−11 STIM001_289 1.419E−09 4.019E−10 2.493E−10 6.900E−10 6.358E−10 3.671E−10 STIM003 3.258E−10 4.769E−10 2.397E−10 3.474E−10 1.201E−10 6.933E−11 STIM001 4.559E−10 2.467E−10 1.467E−10 2.831E−10 1.578E−10 9.112E−11 mICOS N = 1 N = 2 N = 3 Best-Fit values EC50 (M) EC50 (M) EC50 (M) Mean (M) STDEV (M) SEM (M) STIM003_457 7.702E−10 1.353E−09  1.894E−09  1.339E−09 5.621E−10 3.245E−10 STIM001_457 8.627E−10 4.888E−09* 1.077E−08* 8.627E−10 STIM003 5.079E−10 7.544E−10  7.025E−10  6.549E−10 1.299E−10 7.502E−11 STIM001 4.790E−10 5.337E−10  5.786E−10  5.304E−10 4.988E−11 2.880E−11 *EC50 values excluded due to incomplete curve fit.

Example 5c ADCC Assay Using Peripheral Blood Mononuclear Cells (PBMC)

The potential to kill via ADCC (“antibody-dependent cell-mediated cytotoxicity”) of STIM001_289 and STIM003_289 was compared with that of STIM001 and STIM003 in the Delfia BATDA cytotoxicity assay (Perkin Elmer) using human primary NK cells as effector and ICOS-transfected CCRF-CEM cells as target cells. This method is based on loading target cells with an acetoxymethyl ester of fluorescence enhancing ligand (BATDA) which quickly penetrates the cell membrane. Within the cell the ester bonds are hydrolysed to form a hydrophilic ligand (TDA) which no longer passes the membrane. After cytolysis, the ligand is released and can be detected by addition of Europium which forms with the TDA a highly fluorescent and stable chelate (EuTDA). The measured signal correlates directly with the amount of lysed cells.

Isolation of Mononuclear Cells from Human Peripheral Blood:

Leukocyte cones were collected from healthy donors and their content was diluted up to 50 ml with phosphate buffered saline (PBS, from Gibco) and layered into 2 centrifuge tubes on top of 15 mL Ficoll-Paque (from GE Healthcare). PBMC were separated by density gradient centrifugation (400 g for 40 min without brake), transfer in a clean centrifuge tube and then wash with 50 mL PBS, twice by centrifuging at 300 g for 5 min and twice by centrifuging at 200 g for 5 min. PBMC were then resuspended in R10 media (RPMI+10% heat-inactivated Fetal Bovine Serum, both from Gibco) and their cell count and viability assess with EVE™ Automated Cell Counter (from NanoEnTek).

ADCC Assay Methods:

Labelling of target cells was performed according to manufacturer's instruction. Briefly, CCRF-CEM cells were resuspended at 1×10⁶/mL in assay media (RPMI+10% ultra-low IgG FBS, from Gibco) and loaded with 5 μl/mL of Delfia BATDA reagent (Perkin Elmer) for 30 min at 37° C. Cells were then washed 3 times with 50 mL PBS (300 g for 5 min) and resuspended at 8×10⁵/ml (3×) in assay media.

STIM001_289, STIM003_289, STIM001, STIM003 and their isotype controls, IgG1_289 and IgG1 were serially diluted 1:4 in assay media to give final 3× antibody concentrations ranging from 30 nM to 0.12 pM (10-point curve).

NK cells were negatively isolated from PBMC using the EasySep Human NK Cell Isolation Kit (from Stemcell Technologies) and resuspended at 4×10⁶/ml (3×) in assay media.

BATDA-loaded CCRF-CEM and primary NK cells were co-cultured for 4-hours at 37° C. and 5% CO2 at a 5:1 Effector:Target ratio in assay media in the presence of the antibodies under investigation (from 10 nM to 0.04 pM final concentration). Wells containing CCRF-CEM cells only or CCRF-CEM+Delfia lysis buffer (Perkin Elmer) were used to determine spontaneous and 100% BATDA release, respectively. Cell-free supernatants were then transferred into a DELFIA Microtitration Plates and incubated for 15 min at Room Temperature with the Delfia Europium solution (Perkin Elmer). Fluorescent signal at 615 nM was then quantified with Envision Multilabel Reader (PerkinElmer).

Specific dye release induced by the Abs was calculated as: [(Experimental release−Spontaneous release)/(Maximum release−Spontaneous release)]*100.

This experiment was repeated with NK-cells from 2 independent donors and 3 technical replicates were included for each assay condition.

Results:

The ability of STIM001_289 and STIM003_289 bispecific antibodies to induce ADCC was assessed using primary NK cells from 3 independent donors, as effector cells and ICOS-transfected CCRF-CEM as target cells. STIM001 and STIM003 and the relevant isotype controls (IgG1 and IgG1_289) were run in the same experiments. Target cells were loaded with BADTA dye and incubated for 4 hrs either alone (spontaneous release), with lysis buffer (100% release) or with NK cells plus increasing concentration of antibody. Dye release correlates directly with the number of lysed cells. Data are showed as % of specific dye release and plotted against the log of antibody concentrations (FIG. 9 ). In all donors, STIM001_289, STIM003_289, STIM001 and STIM003 increased the % of specific dye release in a concentration dependent manner. Non-linear regression curves (variable slope, 4-parameter) were extrapolated from the data obtained. These data demonstrate that the bispecific constructs have the ability to kill ICOS positive cells in a primary NK dependent ADCC assay. The EC50s of the bispecific antibodies in the ADCC assay were comparable to those of STIM001 and STIM003 monoclonal IgG1 antibodies. See Table E5-2 and FIG. 9 A-C.

TABLE E5-2 EC50 (pM) of STIM001_289 and STIM003_289 antibodies in the ADCC assay using ICOS-transfected CCRF-CEM cells and freshly isolated NK cells as effector cells (non-linear fit, 4-parameters, n = 2). EC50 (pM) Donor 326 Donor 334 Donor 341 Median SD STIM001 42.6 20.5 7.6 23.6 17.7 STIM001_289 30.9 4.1 ~10.3 17.5 19.0 STIM003 21.2 3.7 6.7 10.5 9.4 STIM003_289 11.1 18.3 6.6 12.0 5.9

Example 6 Ability of ICOS/PD-L1 Bispecific Antibody to Neutralize ICOS Binding to ICOS Ligand

Anti-ICOS antibodies in monoclonal and mAb² format were assessed for ICOS ligand (B7-H2) neutralisation using HTRF. These antibodies are capable of neutralising both human and mouse ICOS B7-H2 ligand and were assessed in both Human ICOS Receptor/Human Ligand and Mouse ICOS Receptor/Mouse Ligand HTRF based Neutralisation assays.

Method:

Antibodies were diluted in assay buffer (0.53M Potassium Fluoride (KF), 0.1% Bovine Serum Albumin (BSA) in 1×PBS) from a starting working concentration of either 1 μM or 0.4 μM and serially diluted 1 in 3 over 11 points. 5 μl of titrated antibody were added to 384w solid white assay plate (Greiner Bio-One). Positive and negative control wells received 5 μl of assay buffer only.

5 μl of human ICOS-mFc (Chimerigen) at 20 nM (5 nM final) or 5 ul of mouse ICOS-mFc (Chimerigen) at 4 nM (1 nM final) were added to relevant assay wells. Plate was incubated for 1 hour (hr) at room temperature (RT).

After incubation, 5 μl of ICOS ligand, (B7-H2, R&D Systems) conjugated to Alexa 647 (Innova Bioscience) was diluted to either 14.08 nM (3.52 nM final) for human B7-H2 or 36.08 nM (9.02 nM final) for mouse B7-H2 and added to all wells of assay plate except negative control wells which instead received 5 ul of assay buffer.

Finally, 5 μl of 4.32 nM anti-mouse IgG donor mAb (Southern Biotech) labelled with europium cryptate (Cis Bio), was added to each well and the assay was left in the dark at RT to incubate for a further 2 hours. After incubation, assay was read on Envision plate reader (Perkin Elmer) using a standard HTRF protocol. 620 nm and 665 nm channel values were exported to Microsoft Excel and % Delta-F and % Neutralisation calculations performed. Titration curves and IC50 values [M] were plotted using Graphpad (Prism). IC50 values were calculated by first transforming the data using equation X=Log(X). The transformed data was then fitted using nonlinear regression, using fitting algorithm, log (inhibitor) vs. response—variable slope (four parameters).

Delta-F Calculation

665/620 nm ratio for ratio metric data reduction.

${\%{Delta}F} = {\frac{\left( {665/620{nm}{Well}{Signal}{Ratio} - {Signal}{Negative}{Control}} \right)}{\left( {{Signal}{Negative}{Control}} \right)}*100}$

Signal Negative control=average of minimum signal ratio.

Neutralisation

${\%{Max}({neutralisation})} = {\frac{\left( {\%{Delta} - F{of}{sample}{well} - {Negative}{Control}} \right)}{\left( {{Positive}{Control} - {Negative}{Control}} \right)}*100}$

Results:

In the human ICOS ligand neutralisation system, STIM003_289 and STIM003_457 produced similar IC50 values to STIM003 (mean IC50 values, 0.81±0.28 nM, 0.56±0.18 nM and 0.53±0.15 nM respectively).

In the human ICOS ligand neutralisation system, STIM001_289 and STIM001_457 produced similar IC50 values to STIM001 (mean IC50 values, 2.0±1.7 nM, 1.6±6.9 nM and 1.5±0.75 nM respectively).

In the mouse ICOS ligand neutralisation system, STIM003_289 and STIM003_457 produced similar IC50 values to STIM003 (mean IC50 values, 0.14±0.037 nM, 0.11±0.027 nM and 0.12±0.027 nM and respectively).

In the mouse ICOS ligand neutralisation system, STIM001_289 and STIM001_457 produced similar IC50 values to STIM001 (mean IC50 values, 4.8±1.7 nM, 5.16±1.5 nM and 8.7±6.6 nM and respectively).

In the human ICOS ligand neutralisation system, STIM003 produced similar IC50 values to STIM001 (mean IC50 values, 0.53±0.15 nM, 1.5±0.75 nM respectively).

In the mouse ICOS ligand neutralisation system, STIM003 produced more potent IC50 values to STIM001 (mean IC50 values, 0.12±0.027 nM and 8.7±0.66 nM respectively).

Data are summarised in Table E6-1 and Table E6-2 and FIGS. 10A and B.

TABLE E6-1 IC50 Human ICOS Receptor/ Human H7-H2 Average SD Ab n1 (nM) n2 (nM) n3 (nM) IC50 (nM) (nM) STIM003_289 0.58 0.73 1.11 0.81 0.27 STIM001_289 1.00 0.94 3.95 1.96 1.72 STIM003_457 0.44 0.47 0.77 0.56 0.18 STIM001_457 1.10 1.32 2.38 1.60 0.68 STIM003 0.43 0.45 0.71 0.53 0.16 STIM001 0.96 1.12 2.33 1.47 0.75

TABLE E6-2 IC50 Mouse ICOS Receptor/ Mouse B7-H2 Average SD Patent Ab n1 (nM) n2 (nM) n3 (nM) IC50 (nM) (nM) STIM003_289 0.10 0.17 0.13 0.13 0.04 STIM001_289 3.25 6.53 4.57 4.78 1.65 STIM003_457 0.087 0.13 0.10 0.11 0.02 STIM001_457 3.39 5.49 6.32 5.07 1.51 STIM003 0.87 0.14 0.11 0.37 0.43 STIM001 7.98 8.91 9.25 8.71 0.66

Example 7 Ability of ICOS/PD-L1 Bispecific Antibody to Neutralize PD-L1 Binding to PD1 or CD80 Neutralisation of Human PD-L1 Binding

Bispecific mAb² antibodies STIM001_289 and STIM003_289, two anti-PD-L1 antibodies AbW and AbV, and one isotype control (IgG1_289), were assessed for ability to neutralize human PD-L1 binding to its receptors human PD1 and human CD80 on CHO cells, using flow cytometry (FACS). Ability of the mAb² to neutralize binding of human PD-L1 to its receptors was confirmed in this assay.

Method:

CHO-S cells untransfected (referred to as WT) or transfected with human PD-L1 were diluted in FACS buffer (PBS+1% w/v BSA+0.1% w/v sodium azide) and were distributed to a 96-well V-bottom plate (Greiner) at a density of 5×10⁴ cells per well. Biotinylated human CD80-Fc (R&D Systems) or PD-1-Fc were prepared as a standard curve titration from 1 pM final assay concentration (FAC), 1/3 dilution series in FACS buffer. Antibody and mAb² titrations were prepared from 396 nM working concentration, 198 nM FAC, as a 1/3 dilution series in FACS buffer. Biotinylated PD-1 or CD80 were diluted in FACS buffer to 80 nM working concentration, 40 nM FAC. Plates were centrifuged at 300×g for 3 minutes to supernatant aspirated. 25 μL receptor and 25 μL mAb² solution (or 50 μL of receptor standard curve titration) were added to cells and incubated at 4° C. for 1 hour. Cells were washed with 150 μL of PBS and centrifuged at 300 g for 3 minutes. Supernatant was aspirated and 150 μL PBS added. This wash step was repeated. Presence of bound CD80 or PD-1 was detected by addition of 50 μL of streptavidin-AlexaFluor 647 (Jackson ImmunoResearch) diluted 1/500 in FACS buffer to each well. Cells were incubated 1 hr at 4° C. in the dark. Cells were washed as previously described. To fix cells, 100 μL 4% v/v paraformaldehyde was added and cells incubated for 20 minutes at 4° C., cells were pelleted by centrifugation at 300×g and the plates resuspended in 100 μL FACS buffer. AlexaFluor 647 signal intensity (geometric mean) was measured by flow cytometry using a Beckman Coulter CytoFLEX.

Equation X: Percentage of receptor binding (flow cytometry): Based on geometric mean fluorescence

${\%{of}{specific}{binding}} = {\frac{{sample}{value} - {non} - {specific}{binding}}{{total}{binding} - {non} - {specific}{binding}} \times 100}$

Total binding=biotinylated PD-1 or CD80 only (Isotype antibody at 198 nM) Non-specific binding=mAb² at concentration of 198 nM

Results:

Data are shown in Tables E7-1 and E7-2 below and in FIGS. 11A and B.

TABLE E7-1 IC50 (nM) values for human PD1/PD-L1 neutralisation, detected with streptavidin-AlexaFluor 647. Not calculated: Ab was used in assay with no complete IC50 curve being calculable. See FIG. 11 A. IC50 Human PD1/PD-L1 Neutralisation (nM) STIM003 Not Calculated STIM003_289 0.28 AbW 0.70 STIM001 Not Calculated STIM001_289 0.47 AbV 0.86 IgG1 Not Calculated IgG1_289 0.34

TABLE E7-2 IC50 (nM) values for human CD80/PD-L1 neutralisation, detected with streptavidin-AlexaFluor 647. Not calculated: Ab was used in assay with no complete IC50 curve being calculable. See FIG. 11 B. IC50 Human CD80/PD-L1 Neutralisation (nM) STIM003 Not Calculated STIM003_289 0.26 AbW 0.74 STIM001 Not Calculated STIM001_289 0.43 AbV 0.85 IgG1 Not Calculated IgG1_289 0.21

Neutralisation of Mouse PD-L1 Binding

Ability of the ICOS/PD-L1 bispecific antibody to neutralize binding of mouse PD-L1 to its receptors was confirmed in this assay.

Method:

CHO-S cells untransfected (referred to as WT) or transfected with mouse PD-L1 were diluted in FACS buffer (PBS+1% w/v BSA+0.1% w/v sodium azide) and were distributed to a 96-well V-bottom plate (Greiner) at a density of 5×104 cells per well. Biotinylated mouse PD-1-Fc (R&D Systems) or CD80-Fc (R&D Systems) were prepared as a standard curve titration from 1 pM final assay concentration (FAC), 1/3 dilution series in FACS buffer. Antibody and mAb² titrations were prepared from 44 nM working concentration, 22 nM FAC, as a 1/3 dilution series in FACS buffer. Biotinylated PD-1 or CD80 were diluted in FACS buffer to 80 nM working concentration, 40 nM FAC. Plates were centrifuged at 300×g for 3 minutes to supernatant aspirated. 25 μL receptor and 25 μL mAb² solution (or 50 μL of receptor standard curve titration) were added to cells and incubated at 4° C. for 1 hour. Cells were washed with 150 μL of PBS and centrifuged at 300 g for 3 minutes. Supernatant was aspirated and 150 μL PBS added. This wash step was repeated. Presence of bound CD80 or PD-1 was detected by addition of 50 μL of streptavidin-AlexaFluor 647 (Jackson ImmunoResearch) diluted 1/500 in FACS buffer to each well. Cells were incubated 1 hr at 4° C. in the dark. Cells were washed as previously described. To fix cells, 100 μL 4% v/v paraformaldehyde was added and cells incubated for 20 minutes at 4° C., cells were pelleted by centrifugation at 300×g and the plates resuspended in 100 μL FACS buffer. AlexaFluor 647 signal intensity (geometric mean) was measured by flow cytometry using a Beckman Coulter CytoFLEX.

Equation X: Percentage of receptor binding (flow cytometry)

Based on geometric mean fluorescence

${\%{of}{specific}{binding}} = {\frac{{sample}{value} - {non} - {specific}{binding}}{{total}{binding} - {non} - {specific}{binding}} \times 100}$

Total binding=biotinylated PD-1 or CD80 only (Isotype antibody at 22 nM FAC)

Non-specific binding=mAb² at concentration of 22 nM FAC

Results:

The bispecific mAb² s and an isotype control antibody IgG1_438 were assessed for ability to neutralize binding of mouse PD-L1 to its receptors mouse PD1 and mouse CD80 using FACS. Results are shown in Table E7-3, Table E7-4 and FIG. 12 .

STIM001_457, STIM003_457 and IgG1_438 produced similar IC50s (0.35±0.09 nM, 0.40±0.13 nM, and 0.34±0.05 nM respectively) using the PD-L1 and PD1 neutralising system. These mAb² s also neutralised PD-L1 binding to CD80 (IC50, 0.9±0.03 nM, 0.29±0.0002 nM and 0.27±0.06 nM respectively).

Monoclonal antibodies STIM001, STIM003 and IgG1 control did not neutralize mouse PD-L1 binding to mouse PD1 or mouse CD80.

Example 8 Effect of ICOS/PD-L1 Bispecific Antibody on T Cells in ICOS-Dependent Activation Assay

The agonistic potentials of STIM001_289 and STIM003_289 bispecific antibodies in this assay were compared with those of STIM001 and STIM003 monoclonal antibodies in a human primary T-cell activation assay where anti-CD3 and anti-CD28 antibodies were added concurrently to induce ICOS expression on effector T-cells. Effect of the ICOS co-stimulation on the level of IFN-γ produced by these activated T-cells were assessed using ELISA at 72 hrs post-activation. This assay is used to confirm activity of the ICOS-binding portion of the bispecific antibody. Retention of ability to induce ICOS-mediated T cell activation was confirmed for anti-ICOS antibodies STIM001 and STIM003 in the ICOS/PD-L1 mAb² bispecific format in which the Fcab region binds human PD-L1.

Methods:

PBMC were isolated from human peripheral blood as described in Example 5c and stored in nitrogen for further utilisation.

STIM001_289, STIM003_289, STIM001, STIM003 and their isotype controls, IgG1_289 and IgG1 were serially diluted 1:3 in PBS to give final antibody concentrations ranging from 10 pM to 40 nM (6-point curve). 100 μL of diluted antibodies were coated in duplicate into a 96-well, high-binding, flat-bottom plate (Corning EIA/RIA plate) overnight at 4° C. Plate was then washed with PBS. In some experiments an anti-PD-L1 antibody, AbV, was added at the same concentrations.

T-cells were negatively isolated from frozen PBMC using the EasySep Human T Cell Isolation Kit (from Stemcell Technologies) and resuspended at 2×10⁶/ml in R10 media supplemented with 40 μl/ml of Dynabeads Human T-Activator CD3/CD28 (from Life Technologies).

T-cell suspensions were added to antibody-coated plates to give a final cell concentration of 1×10⁶ cells/ml and cultured for 72 hrs at 37° C. and 5% CO2. Cell free supernatants were then collected and kept at minus 20° C. until analysis of secreted IFN-γ by ELISA with the R&D Systems™ Human IFNγ Duoset® ELISA, using DELFIA® Eu-N1 Streptavidin detection.

This experiment was repeated on T-cells isolated from 4 independent donors and 2 technical replicates were included for each assay condition.

Results:

The levels of IFN-γ induced by STIM001_289, STIM003_289, STIM001, STIM003 and their isotype controls (IgG1_289 and IgG1) were measured in assay replicates and plotted as mean value±Standard Deviation (SD) against the log of antibody concentrations, as showed for one donor (FIG. 13 , A and B). All antibodies increased the levels of IFN-γ in a concentration dependent manner. In some experiments, anti-PD-L1 AbV was added as the same concentration and did not modify the levels of IFN-γ. Non-linear regression curves (variable slope, 4-parameter) were extrapolated from data obtained with 4 independent donors (EC50 values in Table E8). Both STIM001_289 and STIM003_289 increased the IFN-γ levels to the same extent as STIM001 and STIM003 respectively. Both EC50 values (Table E8) and levels of IFN-γ at the plateau (3.3 μM, FIG. 13 C) were comparable for STIM001 vs STIM001_289 and for STIM003 vs STIM003_289 (median values). These data demonstrate that the ICOS binding sites of the anti-ICOS monoclonal antibodies have conserved their ICOS agonistic effect on human primary T-cells when included in the ICOS/PD-L1 bispecific antibodies.

TABLE E8 T-cells from donors EC50 (nM) 278 276 289 295 Median SD STIM001 ~10.2 ~9.9 ~11.2 14.5 10.7 2.11 STIM001-289 8.9 ~10 7.1 11.9 9.45 2.01 STIM003 3.8 7.1 ~4 ~11.6 5.55 3.64 STIM003-289 ~3.8 ~6.0 10.2 12.7 8.1 4.02 ~value corresponding to the best fit found by GraphPad Prism Concentration response curves of IFN-γ following antibody treatments (non-linear fit, 4-parameters, n = 4).

Example 9 Effect of ICOS/PD-L1 Bispecific Antibody on T Cell Activation in Monocyte Co-Culture Primary Cell Assay

This assay can be used to assess ability of the PD-L1 binding site of the bispecific antibody to block PD-L1 PD1 interaction and thereby promote activation of T cells in autologous co-culture of T and B lymphocytes from peripheral blood samples. The effects of STIM001_289, STIM003_289 and IgG1_289 mAb² antibodies on IFN-γ production were compared to those of anti-PD-L1 AbV, STIM003 and IgG1 monoclonal antibodies in a co-culture of purified peripheral blood monocytes and CD45RO+ memory T-cells from the same donor. These cultures were done in the presence of anti-CD3 antibody to provide TCR stimulation. Retention of ability to promote T cell activation in this assay was confirmed for STIM001 and STIM003 in the ICOS/PD-L1 mAb² bispecific format in which the Fcab region binds human PD-L1. The control PD-L1 mAb², lacking an ICOS binding site, was inactive in this assay.

Methods:

PBMC were isolated from human peripheral blood as described in Example 5c and stored in nitrogen for further utilisation.

STIM001_289, STIM003_289, IgG1_289, anti-PD-L1 AbV, STIM003 and their isotype control IgG1 were serially diluted 1:4 in R10 media to give final 4× antibody concentrations ranging from 40 nM to 40 pM (6-point curve). Anti-human CD3 (clone UCHT1 from eBioscience) was diluted in R10 media to a 4×Ab concentration of 2 μg/ml. Monocytes and memory cells were isolated from frozen PBMC from the same donor.

Monocytes were negatively isolated using the Pan Human Monocyte Isolation Kit (Miltenyi biotec) and resuspended at 2×10⁶/ml (4×) in R10 media. CD45RO+ T-cells were isolated by a first round of negative selection for CD3+ T cells (Pan T-cell isolation kit, Miltenyi Biotec), followed by a positive selection for CD45RO+ cells (Human CD45RO MicroBeads, Miltenyi Biotec). CD45RO+ T-cells were then resuspended at 2×10⁶/ml (4×) in R10 media. Cell subsets were co-cultured for 4-days at 37° C. and 5% CO2 at a 1:1 ratio in R10 media in the presence of anti-CD3 (0.5 μg/ml final concentration) and the antibodies under investigation (from 10 nM to 10 pM final concentration). In some wells, no antibody under investigation was added to be able to quantify the basal IFN-γ level (Monocytes+T-cells+CD3 only). After 4-days culture, cell-free supernatants were analysed for IFN-γ release with the R&D Systems™ Human IFNγ Duoset® ELISA, using DELFIA® Eu-N1 Streptavidin detection. Fold increase in IFN-γ was calculated as: (Experimental IFN-γ level/Basal IFN-γ level) This experiment was repeated with monocytes and T-cells isolated from 7 independent donors and 3 to 5 technical replicates were included for each assay condition (dependent of the number of cells available).

Results:

The levels of IFN-γ induced by STIM001_289, STIM003_289, IgG1_289, STIM003, PD-L1 AbV and their isotype control (IgG1) were measured in assay replicates and plotted as mean value±standard deviation (SD) against the log of antibody concentrations, as shown for one donor (FIG. 14 ). Unlike STIM003, the antibodies binding PD-L1 such as STIM001_289, STIM003_289, IgG1_289 and anti-PD-L1 AbV all increased the levels of IFN-γ in a concentration dependent manner. This experiment was repeated in 7 independent donors and high variability in IFN-γ production was noticed in absence of antibody under investigation: 311±177 μg/ml (mean±SD, n=7). Levels of IFN-γ obtained were then normalized by the production of IFN-γ in absence of antibody under investigation. The maximum increase in IFN-γ (values at 10 nM) for all 7 donors was plotted and analysed using Friedman statistic test (FIG. 14 ). The mean increase in IFN-γ level compared to the isotype control (IgG1) induced by was significant for anti-PD-L1 AbV, STIM003_289 and STIM001_289 and was close to significance for IgG1_289 which also binds to PD-L1. Altogether this data is confirming that the PD-L1 binding sites in the bispecific antibodies retain ability to block PD-1/PD-L1 interaction and can subsequently activate T cells.

Example 10 Anti-Tumour Efficacy of ICOS/PD-L1 Bispecific Antibody on J558 Myeloma in Vivo

The J558 syngeneic tumour model was used to assess the effect of bispecific anti-ICOS/anti-PD-L1 antibodies on myeloma. Two ICOS/PD-L1 mAb² bispecific antibodies, STIM001_457 and STIM003_457, were tested in Balb/c mice using the sub-cutaneous J558 plasmacytoma:myeloma cell line (ATCC, TIB-6), to determine how STIM001_457 hIgG1 and STIM003_457 hIgG1 affect the growth of the tumour.

Method:

Balb/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 5×10⁶ cells (passage number below P15) were subcutaneously injected (in 100 μl) into the right flanks of mice. Unless stated otherwise, on day 11 post tumour cells injection, the animals were randomised based on tumour size and treatments were initiated. The J558 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in DMEM supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 90% at the time of tumour cell injection.

Treatment was initiated when the tumours reached an average volume of ˜140 mm{circumflex over ( )}3. Animals were then allocated to 3 groups with similar average tumour size (see Table E10-1 below for the dosing groups). Both bispecific antibodies recognise mouse ICOS (Fab portion) and mouse PD-L1 (Fcab portion) and were dosed IP (dosed at 200 ug per dose) from day 11 (post tumour cell implantation) twice a week for 3 weeks unless the animals had to be removed from study due to welfare (rare) or tumour size. As a control, a group of animals (n=10) was dosed at the same time using a saline solution. Tumour growth was monitored over 37 days and compared to tumours of animals treated with saline. Animal weight and tumour volume were measured 3 times per week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on studies until their tumour reached an average diameter of 12 mm3 or, rarely, when incidence of tumour ulceration was observed (welfare).

TABLE E10-1 Treatment groups for the J558 efficacy study. Number of Treatment regimen twice per week Groups animals from day 11 (Mon/Fri) 1 10  Saline 2 8 STIM003_457 200 ug per dose 3 8 STIM001_457 200 ug per dose

Results:

The J558 syngeneic model is highly aggressive. All animals in the saline control group (n=10) had to be removed from studies by day 21 due to tumour size. However, both bispecific antibodies STIM001_457 and STIM003_457 demonstrated good efficacy when used as the sole therapy in this model with, respectively, 50% and 62.5% of the animals cured from their disease by day 37. The anti-tumour efficacy of both bispecific antibodies resulted in improved overall survival of the treated animals (time on study) which was significant vs saline treated group for both antibodies (p<0.05 for STIM003_457 and p<0.001 for STIM001_457). See FIG. 15 and FIG. 16 . Table E10-2 below shows hazard ratio (logrank) and p value for the different treatment comparisons.

TABLE E10-2 P value (Log Rank, Hazard Ratio Conditions Mantel-Cox) (logrank) Saline vs STIM003_457 P < 0.05  2.622 Saline vs STIM001_457 P < 0.001 5.011 STIM003_457 vs STIM001_457 Not Significant 1.498

Example 11a Anti-Tumour Efficacy of ICOS/PD-L1 Bispecific Antibody on CT26 Tumours in Vivo

This example demonstrates strong anti-tumour efficacy in vivo in a CT-26 syngeneic model by co-targeting ICOS and PD-L1 using a bispecific antibody. Bispecific mAb² TIM001_457 hIgG1 and STIM003_457 hIgG1 were both effective in this study.

Method:

Efficacy studies were performed in BALB/c mice using the sub-cutaneous CT-26 colon carcinoma model (ATCC, CRL-2638). BALB/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 1×10E5 CT-26 cells (passage number below P20) were subcutaneously injected into the right flanks of mice. Unless stated otherwise, treatments were initiated at day 6 post tumour cells injection. The CT-26 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in RPMI supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 90% at the time of tumour cell injection.

STIM001_457 or STIM003_457 bispecific mAb² antibody was each used as the sole therapeutic agent. These antibodies bind to mouse ICOS via the Fab domains and to mouse PD-L1 via the Fc domain (Fcab). Bispecific antibodies were dosed intraperitoneal (IP) at 200 μg each (1 mg/ml in 0.9% saline) three times per week from day 6 (dosing for 2 weeks between day 6-17) post tumour cell implantation. Tumour growth was monitored and compared with tumours of animals in a saline-treated control group and with an isotype IgG1_457 control mAb² antibody which binds to mouse PD-L1 but does not bind to ICOS. Animal weight and tumour volume were measured 3 times per week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on studies until their tumour reached an average diameter of 12 mm3 or, rarely, when incidence of tumour ulceration was observed (welfare). Mice were re-challenged at day 50. The humane endpoint survival statistics were calculated using the Kaplan-Meier method with Prism. This approach was used to determine if specific treatments were associated with improved survival.

TABLE 11a-1 Bispecific antibody treatment groups in the CT26 model. Number of Treatment regimen (dosed I.P. 3 time a Groups animals week from day 6 for 2 weeks) 1 10 Saline 2 10 IgG1_457 Fc effector-disabled (LAGA) control 200 μg per dose 3 10 STIM003_457 200 μg per dose 4 10 STIM001_457 200 μg per dose

Results:

The present experiment clearly demonstrates that both bispecific antibodies significantly delayed tumour growth and extended the survival (time to reach humane endpoint/time on study) of treated animals when compared to saline or IgG1_487 LAGA treated animals. When administered individually as monotherapies rather than in combination, anti-ICOS and anti-PD-L1 antibodies were each significantly less effective at preventing CT26 tumours compared with the bispecific antibodies (data not shown).

In this experiment STIM003_457 antibody was more effective at inhibiting tumour growth than STIM001_457. STIM003_457 demonstrated the strongest anti-tumour efficacy and improved survival (60% were cured from the disease at day 50) whereas STIM001_457 resulted in 3 out 10 animals (30%) with no sign of disease at the end of the study (day 50). See FIG. 17 .

The humane endpoint survival statistics were calculated using the Kaplan-Meier method with Prism. This approach was used to determine if specific treatments were associated with improved survival. See FIG. 18 .

TABLE 11a-2 P value Log-rank Hazard Ratio (Mantel- conditions (A vs B) (Mantel-Cox) test Haenszel A vs B) Saline vs STIM003_457 P < 0.0001 19.71 saline vs STIM001_457 P < 0.0001 17.55 STIM003_457 vs 0.2831 0.5155 STIM001_457 (not significant)

These data confirm that co-targeting ICOS and PD-L1 using a bispecific antibody, even as the sole treatment agent, is effective to trigger an anti-tumour response in the CT26 model.

Example 11b Treatment with Bispecific ICOS/PD-L1 Antibody Induces Long-Term Immune Memory to Tumour Antigens

Animals that showed full tumour regression following treatment with the bispecific antibodies in Example 11a were challenged again with tumour cells. Of a total of 9 animals, 5 were re-challenged with CT26 to determine whether the animals' immune system showed a memory response to CT26 cells that could prevent these tumours from growing post re-challenge implantation. In addition, 4 animals were challenged with implanted EMT-6 tumour cells, which their immune systems had not been exposed to previously.

TABLE 11b Bispecific antibody re-challenge groups Animal ID Previous treatment Cells implanted number groups (re-challenge) CB43 STIM003_457 CT-26 FCA5 STIM003_457 CT-26 1147 STIM003_457 CT-26 FEDD STIM001_457 CT-26 A6E7 STIM001_457 CT-26 E692 STIM003_457 EMT-6 C687 STIM003_457 EMT-6 D063 STIM003_457 EMT-6 FC52 STIM001_457 EMT-6

Results are shown in FIG. 19 . All the animals that had rejected the CT-26 tumours in response to one of the two ICOS/PD-L1 bispecifics managed to reject the newly-injected CT-26 cells in the absence of further treatment. On the other hand, animals that were injected with the new cell line EMT-6, all quickly demonstrated the presence of an established EMT-6 tumour. Altogether the data demonstrate that animals that previously rejected CT-26 tumour in response to either of the bispecific antibody treatments were fully resistant to the CT-26 tumour but not to the unrelated EMT6 tumour. This result indicates that the mice cured by the bispecific antibody therapy had established a long-term memory to tumour antigens expressed specifically by the CT-26 tumour.

Example 12 Anti-Tumour Efficacy of ICOS-PD-L1 Bispecific Antibody on A20 Tumours In Vivo

ICOS/PD-L1 bispecific antibodies STIM001_457 and STIM003_457 showed strong anti-tumour efficacy in vivo in the A20 syngeneic model when used as sole therapy.

Method:

Efficacy studies were performed in BALB/c mice using the sub-cutaneous A20 Reticulum Cell Sarcoma model (ATCC number CRL-TIB-208). BALB/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 5×10E5 A20 cells (passage number below P20) were subcutaneously injected into the right flanks of mice. Unless stated otherwise, treatment was initiated at day 8 post tumour cells injection. The A20 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in RPMI supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 85% at the time of tumour cell injection.

The antibodies were dosed intraperitoneally (IP) at 200 μg each (1 mg/ml in 0.9% saline) twice per week from day 8 (dosing for 3 weeks between day 8-25, six doses in total) post tumour cell implantation. Tumour growth was monitored and compared to tumours of animals treated with a IgG2a isotype control group and IgG1_457 (anti-PD-L1 control). Animal weight and tumour volume were measured 3 times a week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width²). Mice were kept on study until their tumour reached an average diameter of 12 mm³ or, rarely, when incidence of tumour ulceration was observed (welfare).

TABLE E12-1 Bispecific antibody treatment groups. Groups Number of animals Treatment regimen 1 10 IgG2a isotype control 2 10 IgG1_457 3 10 STIM003_457 4 10 STIM001_457

Results:

The STIM001_457 and STIM003_457 bispecific antibodies significantly delayed the growth of A20 sub-cutaneous tumours and resulted in extended survival (time to reach humane endpoint) of the treated animals when compared to IgG2a isotype control or IgG1_487 treated animals. All animals in the two control groups had to be removed from the study by day 40. Notably, both bispecific antibodies demonstrated a strong anti-tumour efficacy with 40 and 70% of the animals presented no signs of the disease at day 41. See FIG. 20 and FIG. 21 .

Example 13 Anti-Tumour Efficacy of ICOS/PD-L1 Bispecific Antibody on EMT6 Tumours in Vivo

Anti-tumour in vivo efficacy of co-targeting ICOS and PD-L1 with bispecific antibodies was assessed in an EMT-6 syngeneic model using STIM001_457 and STIM003_457.

Method:

Efficacy studies were performed in BALB/c mice using the sub-cutaneous EMT-6 breast carcinoma model (ATCC number CRL-2755). BALB/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 2.5×10E5 EMT-6 cells (passage number below P20) were subcutaneously injected into the right flanks of mice. Unless stated otherwise, treatments were initiated at day 6 post tumour cells injection. The EMT-6 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in Waymouths MB 752/1 with 2 mM L-glutamine and supplemented with 15% foetal calf serum. Cell viability was confirmed to be above 90% at the time of tumour cell injection.

STIM001_457 and STIM003_457 were each used as single therapeutic agents, dosed intraperitoneally (IP) at 200 μg (1 mg/ml in 0.9% saline) twice a week from day 6 (dosing for 3 weeks between day 6-23) post tumour cell implantation. Tumour growth was monitored and compared to tumours of control animals treated with a saline and IgG1_457 LAGA control. IgG1_457 LAGA can bind PD-L1 and block PD1-PD-L1 interaction but does not bind to ICOS. Animal weight and tumour volume were measured 3 times per week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on studies until their tumour reached an average diameter of 12 mm3 or, rarely, when incidence of tumour ulceration was observed (welfare).

TABLE E13-1 Bispecific antibody treatment groups. Groups Number of animals Treatment regimen 1 10 Saline 7 10 IgG1_457 Fc effector-disabled (LAGA) mAb² 200 ug per dose 8 10 STIM003_457 200 ug per dose 9 10 STIM001_457 200 ug per dose

Results:

Data are shown in FIG. 22 and FIG. 23 , and in Table E13-2 below.

Both bispecific antibodies significantly delayed EMT6 tumour growth and resulted in a longer survival (time to reach humane endpoint) when compared with animals treated with saline or IgG1_457 LAGA. STIM001_457 was marginally more potent in this model and resulted in the strongest anti-tumour efficacy and improved survival (30% vs 20% were cured from the disease at day 44 for STIM001_457 and STIM003_457, respectively), however this difference is not significant. Interestingly the IgG1_457 LAGA treatment resulted in tumour growth delay (vs saline treated group) but unlike what was observed for the ICOS/PD-L1 bispecific, this efficacy did not result in complete response (i.e., absence of tumour at the end of the experiment).

TABLE E13-2 P value and the hazard ratio for different treatment comparisons. The statistics were calculated with Prism. Hazard Ratio P value Log-rank (Mantel-Haenszel Conditions (A vs B) (Mantel-Cox) test A vs B) Saline vs STIM003_457 0.0304 3.676 200 ug Saline vs STIM001_457 0.0009 8.683 200 ug IgG1_457 LAGA 200 ug vs 0.2641 1.839 STIM003_457 200 ug IgG1_457 LAGA 200 ug vs 0.3174 1.741 STIM001_457 200 ug STIM003_457 200 ug vs 0.9692 1.022 STIM001_457 200 ug

Comparing the ICOS/PD-L1 bispecific mAb² antibodies with two separate monoclonal antibodies (anti-ICOS STIM003 and anti-PD-L1 antibody), the bispecific mAb² antibodies showed efficacy where the combination did not. See FIG. 24 and FIG. 25 .

Example 14 Anti-ICOS Agonism of MJ T Cells in PD-L1-Dependent Assay

In this ICOS-dependent T cell activation assay, the agonistic potentials of STIM001_289 and STIM003_289 bispecific antibodies were compared with those of STIM001 and STIM003 IgG1 monoclonal antibodies as well as the isotype controls of both the bispecific and monoclonal antibodies in a cell stimulation assay. ICOS expressing MJ cells were stimulated and IFN-γ at 72 hrs post-activation was measured as the readout. This assay can be used to assess the ability of the PD-L1 binding site of the bispecific antibody to present the antibody in a way that promotes activation of target cells through ICOS signalling.

PDL1-dependent release of IFN-γ by the target cells could only be seen with the molecules that bind both ICOS and PDL1 (B7-H1). There was no significant IFN-γ release above background for any of the antibodies when these were added to plates pre-coated with the negative control (BSA). When the positive control Goat anti-human IgG Fcg fragment specific F(ab′)2, which binds the Fc-domains of both the bispecific and the IgG1 monoclonal antibodies, was coated on the plates, all antibodies that bind ICOS, i.e. STIM001_289, STIM003_289, STIM001 IgG1 and STIM003 IgG1 could induce IFN-γ release, whereas the isotype controls could not. When the plates were coated with PDL1-Fc, only STIM001_289 and STIM003_289 were able to induce IFN-γ, whereas the HYB.CTRL_289, which is not able to bind ICOS, did not. Similarly, the IgG1s, which are unable to bind PDL1 did not induce the release of IFN-γ. Altogether, this experiment demonstrates that the bispecific antibodies can induce PDL1-dependent ICOS agonism.

Materials and Methods Plate Coating

96-well, sterile, flat, high binding plates (Costar) were coated in duplicate overnight at 4° C. with 100 μl/well of DPBS (Gibco) containing either 1% w/v of bovine serum albumin (BSA; Sigma) or 10 μg/ml of recombinant human B7-H1-Fc chimera (RnD Systems) or 10 μg/ml goat anti-human IgG Fcg fragment specific F(ab′)2 (Jackson ImmunoResearch). Plates were then washed twice with 200 μl/well of DPBS and blocked with 1% BSA for 1 hr at room temperature (RT). The plates were then washed again twice with 200 μl/well of DPBS before the addition of serial dilutions of antibodies.

Antibody Addition

Serial 1:3 dilutions of STIM001, STIM003 and HYB. CTRL (isotype control) as an IgG1 and STIM001_289, STIM003_289 and HYB. CTRL_289 from 10 μg/ml to 0.51 ng/ml were prepared in 1% BSA/DPBS, added to the plates and agitated for 1.5 hrs at RT. The plates were washed again 2× with PBS before the addition of MJ cells. To account for background, several wells of the plate were left empty and to enable calculation of percent effect several wells were stimulated with the Cell Stimulation Cocktail ( 1/20×; eBioscience).

Cell Stimulation

MJ [G11] cell line (ATCC© CRL-8294™) was grown in IMDM (Gibco or ATCC) supplemented with 20% heat inactivated FBS. The cells were counted and 10000 cells/well (100 μl/well) of cell suspension was added to the protein coated plates. Cells were cultured in the plates for 3 days at 37° C. and 5% CO₂. Cells were separated from the media by centrifugation and the supernatants collected for IFN-γ content determination.

Measuring IFN-γ Levels

The IFN-γ content in each well was determined using a modification of the Human IFN-gamma DuoSet ELISA kit (R&D systems). Capture antibody (50 μl/well) was coated overnight at 4 μg/ml in DPBS on black flat bottom, high binding plates (Greiner). The wells were washed three times with 200 μl/well of DPBS+0.1% Tween. The wells were blocked with 200 μl/well of 1% BSA in DPBS (w/v), washed three times with 200 μl/well of DPBS+0.1% Tween and then 50 μl/well of either the IFN-γ standard solutions in RPMI or neat cell supernatant were added to each well. The wells were washed three times with 200 μl/well of DPBS+0.1% Tween before adding 50 μl/well of the detection antibody at 200 ng/ml in DPBS+0.1% BSA. The wells were washed three times with 200 μl/well of DPBS+0.1% Tween before adding 50 μl/well of streptavidin-europium (Perkin Elmer) diluted 1:500 in Assay buffer (Perkin Elmer). The wells were washed three times with 200 μl/well of TBS+0.1% Tween before developing the assay by adding 50 μl/well of Delfia enhancement solution (Perkin Elmer) and measuring the fluorescence emitted at 615 nm on the EnVision Multilabel Plate Reader.

Data Analysis

IFN-γ values for each well were extrapolated from the standard curve and the average background levels from media-only wells were subtracted. The percent effect was calculated as the fraction of signal compared to the IFN-γ values obtained from wells stimulated with the Cell Stimulation Cocktail. The percent effect values were then used in GraphPad prism to fit a 4-parameter log-logistic concentration response curve.

Results

FIG. 26 shows a representative example from two independent experiments.

PDL1-dependent release of IFN-γ by ICOS positive cells could only be seen with the molecules that bind both ICOS and PDL1 (B7-H1). There was no significant IFN-γ release above the background for any of the antibodies when these were added to plates pre-coated with the negative control (BSA). When the positive control Goat anti-human IgG Fcg fragment specific F(ab′)2 was coated on the plates, all antibodies that bind ICOS, i.e. STIM001_289, STIM003_289, STIM001 IgG1 and STIM003 IgG1 could induce IFN-γ release, whereas the isotype controls could not. When the plates were coated with PDL1-Fc, only STIM001_289 and STIM003_289 were able to induce IFN-γ, whereas the HYB. CTRL_289, which is not able to bind ICOS, did not. Similarly, the IgG1s, which are unable to bind PDL1 did not induce the release of IFN-γ. Altogether, this experiment demonstrates that the bi-specific antibodies can induce PDL1-dependent ICOS agonism.

Example 15 Formation of Intercellular Bridge by ICOS/PD-L1 Bispecific Antibody

This Example provides data indicating that the mAb2 bispecific antibodies of the invention are able to bridge cells expressing ICOS and PD-L1 respectively, in the manner illustrated in FIG. 1 .

A flow cytometry protocol was developed to assess the ability of the mAb² STIM001_289 and STIM003_289 to promote the bridging of cells expressing ICOS and cells expressing PD-L1. This experiment aimed to demonstrate that the mAb2 can link cells expressing the targets. This data will ultimately be critical to demonstrate that the mAb2 can trigger agonism of ICOS^(+νe) cells through cross-presentation using PD-L1 positive cells (such as antigen presenting cells and tumour cells). This PD-L1 dependent ICOS agonism is expected to be important part of the mechanism of action of the mAb² in PD-L1 rich tumour microenvironment. For this purpose, CHO cells expressing human PD-L1 were stained with CellTrace™ Far Red (Invitrogen C34572) which emits at 661 nm while CHO cells expressing human ICOS were stained with CellTrace™ Violet (Invitrogen C34571) which emits at 450 nm. Stained cells were incubated with a titration of mAb² antibodies or a combination of the parental monospecific antibodies and then processed with a flow cytometer.

Material and Methods

CHO human PD-L1 and CHO human ICOS cells were harvested, counted, washed, and re-suspended in PBS (Gibco 14190169) at 1 million cells per mL. CellTrace™ Far Red and CellTrace™ Violet dyes were diluted 1:2000 and incubated with their respective cells for 20 min at 37° C. in the dark, according to manufacturer recommendations. Buffer (PBS (Gibco 14190169), 1% BSA (Sigma) 0.1% Na Azide (Severn Biotech 40-2010-01)) was then added in excess for an additional 5-minute incubation step. Cells were spun down, re-suspended in the above buffer at 2 million cells per mL and incubated for at least 10 minutes at 37° C. before proceeding with binding protocol. Unstained cells were kept and used to set up the gating strategy.

MAb² STIM001_289 and STIM003_289, human IgG1 and Hybrid_289 were prepared in buffer at 450 nM and diluted as per 1:3 series, 11 points in triplicates. 50 μL of CHO human PD-L1 cells labelled with CellTrace™ Far Red, 50 μL of CHO human ICOS labelled with CellTrace™ Violet and 50 μL of antibody were added to a 96 well V-bottom PS plate (Greiner 651901).

The monospecific antibodies STIM001 and STIM003, as well as the Hybrid_289 were prepared in buffer at 900 nM and diluted as per 1:3 series, 11 points in triplicates. 25 μL of STIM001 or STIM003 were added to 25 μL of Hybrid_289, 50 μL of labelled CHO human PD-L1 and 50 μL of CHO human ICOS in a 96 well V-bottom PS plate.

Assay plates were incubated at room temperature for 1 hour under gentle agitation (450 rpm) before being read using the Attune NxT flow cytometer (Thermo Fisher) for the detection of fluorescence at 661 nm and 450 nm. FCS files were analysed with FlowJo® software V7.00. Single cells and duplets were gated based on the forward and side scatter dot plot.

Results

Dot plots graphs (see FIG. 27 ) resulted in the identification of four different gates: a double negative quadrant corresponding to a very small population (Q4 in FIG. 27 ) of unstained CHO human PD-L1 and unstained CHO human ICOS; two quadrants positive for one of the two fluorophores (either at 661 nm [PD-L1 CHO cells, Q3 in FIG. 27 ] or at 450 nm [ICOS CHO cells, Q1 in FIG. 27 ]); and a quadrant of dual positive staining (at 661 nm and 450 nm, Q2 in FIG. 27 ) composed of stained PD-L1 and ICOS CHO cells. Cells were seen in this quadrant only when the ICOS/PD-L1 mAb² were used. Percentages of double positive cells were plotted into Prism against antibody titrations.

Using as baseline the average percentage of double positive cells obtained with the human IgG1 isotype control, the areas under curve were also calculated for each condition. The highest area under curve was obtained for antibodies able to recruit the most of PD-L1 and ICOS cells and for the widest range of concentrations. See Table E15 and FIG. 28 .

TABLE E15 Area under curve STIM003_289 19.43 STIM001_289 25.45 Hybrid_289 1.22 Human IgG1 0.21 STIM003 + Hybrid_289 2.45 STIM001 + Hybrid_289 0.89 Baseline 2.50 % Double positive Cells

MAb² STIM001_289 and STIM003_289 were able to recruit cells expressing human PD-L1 with cells expressing human ICOS. This data confirmed that the mAb² were able to link/bridge ICOS and PD-L1 positive cells such as effector T cells and APC cells, respectively.

At low concentrations of mAb², there was not enough antibody to bind two cell lines. The maximal percentage of double positive cells was reached when the concentration of mAb² was optimal to recruit the two cell lines (at 1.85 nM for STIM001_289 and at 0.7 nM for STIM003_289). At high concentrations of mAb² antibody, a decrease in the percentage (bell shape curve) of double positive cells was observed. This is expected to be due to saturation of target binding on individual cells by the excess of the antibody. The targets of the CHO human PD-L1 cells and the targets of the CHO human ICOS were both saturated with high mAb² concentration therefore a same mAb² is not able to simultaneously bind two cells.

STIM001_289 could reach the same maximum of double positive cells (˜16%) to STIM003_289, but the double positive signal was observed over a wider range of concentrations for STIM001_289. This was confirmed by a higher area under curve for STIM001_289 than for STIM003_289. This difference may be explained by the different affinities of STIM001 and STIM003 for human ICOS. Altogether, this data confirmed the ability of the mAb² STIM001_289 and STIM003_289 to bridge ICOS and PD-L1 positive cells, such as effector and APC/tumour cells. This demonstrates the potential of triggering PD-L1 dependent cross presentation of the Mab2 to ICOS cells which would be a pre-requisite to PD-L1 dependent ICOS agonism.

Example 16 Pharmacodynamic Study of Tumour and Spleen Treated with STIM003_457 or STIM001_457 in the CT26-WT Model Methods

To ascertain the effects of the bispecific antibodies on certain immune cells in the tumour microenvironment (TME) and peripheral tissue, STIM003_457 and STIM001_457 were given IP twice to CT26-WT tumour-bearing mice. The tumour and spleen were removed for immune cells content analysis by FACS. 24 female BALB/c mice were injected subcutaneously with 0.1×106 cells/mouse of CT26-WT cells, and their tumours allowed to grow. 13 and 15 days post-implantation, mice were dosed intra-peritoneally with either saline, STIM003_457 or STIM001_457 at a fixed dose of 200 μg each. On day 16 post tumour cells-implantation, all mice were culled and tumour, spleen and tumour-draining lymph node (TDLN) were removed for ex vivo analysis. Tumours were dissociated using a mouse tumour dissociation kit (Miltenyi Biotec), followed along with spleen by the MACS gentle dissociator. Spleen cells were incubated for a short period with red blood cell lysis buffer, then all tissues were filtered through 70 μm (tumour) and 40 μm (spleen) cell strainers. The resulting single cell suspensions were washed twice with RPMI+10% FBS complete media, resuspended in FACS buffer and plated into a v-bottomed deep-well 96-well plate. Cells were stained with Live Dead Fixable Yellow viability dye (Life Technologies), followed by washing and an incubation with anti-CD32/CD16 mAb (eBioscience). Afterwards, the following antibodies were added according to three panels: CD3 (17A2), CD45 (30-F11), CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61.5), B220 (RA3-6B2) and ICOS-L (HK5.3), all obtained from eBioscience. Fluorescence-minus-ones were performed in parallel. For staining of intracellular markers (FoxP3), samples were fixed, permeabilized, and stained with FoxP3 mAbs (FJK-16s, eBioscience). Finally, samples were resuspended in PBS and data acquired on the Attune flow cytometer (Invitrogen) and analysed using FlowJo V10 software (Treestar). Gathered data was statistically analysed using the non-parametric Kruskal-Wallis test, followed by post-hoc Dunn's multiple comparisons test (GraphPad Prism V7.0).

STIM003_457 and STIM001_457 Preferentially Deplete TRegs in the TME

STIM003_457 and STIM001_457 significantly depleted T_(Regs) (defined as CD4⁺ CD25⁺ FoxP3⁺ cells) in the TME when compared to saline (FIGS. 29A and B). There is a clear reduction in both the percentage of T_(Regs) in the whole tumour (all cells), and also as a percentage of total CD4⁺ cells. Correspondingly, we saw a significant increase in the effector CD4 and CD8 T cell: T_(Regs) ratios in the tumour. A high effector T cell: T_(Regs) ratio has been previously associated with increased overall survival in cancer patients and is a key indicator of anti-tumour efficacy for immuno-modulatory molecules. FIG. 29C demonstrates the increase in the number of CD4⁺ effector cells (defined as CD4⁺ CD25⁻ FoxP3⁻ cells) compared to T_(Regs) for both bi-specific antibodies. FIG. 29D shows the same increase in the number of CD8⁺ cells compared to T_(Regs). The same cell types were investigated in the spleen and as expected, a high/full depletion of T_(Regs) was not observed.

There was only a marginal but yet significant depletion of TRegs in response to STIM003_457 when compared to saline (when TRegs as a percentage of total live cells in the spleen was considered see FIG. 30A). However no significant difference was seen in either antibody group in TRegs as a percentage of total CD4+ cells (see FIG. 30B), and there was no significant increase in effector cell to TRegs ratio in response to either bi-specific antibodies in the spleen. Furthermore, examination of the same cells in the tumour-draining lymph node (TDLN) yielded similar results, with no significant difference in any group in any test (results not shown). Overall, (from the 3 tissues analysed), it can be concluded that STIM003_457 and STIM001_457-mediated a strong and significant TRegs depletion selectively in the TME, which results in an increase of effector T-cells to TReg ratio.

ICOS-L Expression on B-Cells in the Periphery is Increased

The percentage of B-cells (defined as CD45+ B220+ cells) which expressed ICOS ligand (ICOS-L) in the spleen was determined by FACS for each treatment group. Both anti-ICOS/anti-PDL1 bispecific antibodies significantly increased the percentage of ICOS-L expressing B-cells by more than 30% when compared to the saline group (see FIG. 31A). The mean fluorescence intensity (MFI) which is used as an indication of ICOS-L relative expression on B-cells was also shown to increase significantly in both groups (see FIG. 31B). In both cases, ICOS-L expression was most increased in animals treated with STIM003_457. Increase in the MFI and in the percentage of ICOS-L-expressing cells indicates that not only was ICOS-L expression more widespread within the B-cell population of antibody-treated animals, but also that expression on each cell was upregulated.

Example 17 Strong Anti-Tumour In Vivo Efficacy in CT26 Syngeneic Model by Combining STIM003_574 with Anti-PD1 (RMT1-14) or Anti-CTLA-4 (4F10)

Efficacy studies were performed in BALB/c mice using the sub-cutaneous CT26 colon carcinoma model (ATCC, CRL-2638). BALB/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 1×10E5 CT26 cells (passage number below P20) were subcutaneously injected into the right flanks of mice. All treatments were initiated at day 6 post tumour cells injection. The CT26 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in RPMI supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 90% at the time of tumour cell implantation.

In order to assess how the anti-ICOS/anti-PD-L1 bispecific antibody would combined with an anti-PD1 or an anti-CTLA4, we performed an efficacy study experiment using [STIM003_574 hIgG1+/− the PD1 antibody RMT1-14] and [STIM003_574 hIgG1+/− the CTLA antibody 4F10]. For the in vivo efficacy studies, STIM003_574 bi-specific (which binds both mouse ICOS and mouse PD-L1 proteins) was also compared to the efficacy of a combination between the two mAbs STIM003 mIgG2a and anti-PD-L1 (AbW).

In this experiment the antibodies were administered concomitantly by intraperitoneal (IP) injections. All antibodies were diluted in (1 mg/ml in 0.9% saline) and dosed from day 6 (as shown in table below) three times a week for 2 weeks (between day 6-17) post tumour cell implantation. Tumour growth was monitored and compared to tumours of animals treated with saline. Fixed doses of 200 μg or 60 μg were used which correspond to a dose of 10 mg/kg and 3 mg/kg respectively for mice of 20 g. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width²). Animal weight was also recorded 3 times a week from the day of tumour cell injection. Mice were kept on studies until their tumour reached an average diameter of 12 mm³ or, in rare cases, when incidence of tumour ulceration was observed (welfare). The experiment was stopped at day 46 (40 days after the start of the treatment).

TABLE E17 Treatment groups for CT26 study. Number of Treatments and dose Groups animals (3 times per week for 2 weeks from day 6) 1 8 Saline 2 8 STIM003_574 hIgG1 (200 μg) 3 8 aCTLA4 (4F10) (200 μg) 4 8 aPD-1 (RMT1-14) (200 μg) 5 8 STIM003_574 hIgG1/aCTLA4 (4F10) combo (200 μg each) 6 8 STIM003_574 hIgG1/aPD-1(RMT1-14) combo (200 μg each) 7 8 STIM003 mIgG2a (60 μg) + anti-PD-L1 (AbW) (200 μg)

The combination of antibodies targeting immune checkpoint (e.g. anti-PD1+anti-CTLA-4) is often associated with adverse events due to the strong activation of the immune system. In the present experiment some of the groups were effectively receiving triple combinations (targeting ICOS, PD-L1 and PD1 or CTLA-4). We used the average animal weight for each group as a surrogate of tolerability of the different treatments. As shown in FIG. 32 , we did not observe a decrease in average weight in any of the groups, confirming that these combinations were well tolerated in the animals.

In parallel to monitoring a possible variation in weight, the tumour size for each animal was also measured over 40 days following the start of the treatments (initiated on day 6). As shown in FIG. 33 , animals treated with saline had to be sacrificed by day 20 due to tumour size or in one instance due to ulceration of the tumour. On the other hand, the animals treated with STIM003_574 and the animals treated with a combination of STIM003 mIgG2a and anti-PD-L1 (AbW) demonstrated an anti-tumour response with, respectively, 37.5% and 62.5% of the animals still on study by day 46. Importantly the anti-tumour efficacy that we observed with STIM003_574 was significantly improved by combining the bi-specific with anti-PD1 or with anti-CTLA-4. Anti-PD1 and anti-CTLA4 monotherapies could trigger an anti-tumour response but this was only for a short term. In fact, only 1 and 2 animals from the anti-PD1 and the anti-CTLA-4 monotherapy groups were still on study on day 46. Whereas the combination of STIM003_574 with anti-PD1 or anti-CTLA-4 resulted in 6 and 8 animals respectively still on study by the endpoint day.

The synergy observed with the combination of the anti-ICOS/anti-PD-L1 bispecific antibody with anti-CTLA4 was reproduced using another antibody, 9D9. This antibody has depletion potential and is therefore considered to be a close surrogate of the FDA-approved anti-CTLA-4 antibody ipilimumab. In this further study, groups of 8 CT26 tumour-bearing mice were treated with (i) saline (negative control), (ii) 200 μg anti-CTLA-4 antibody 9D9, (iii) 200 μg STIM003_574 or (iv) 200 μg STIM003_574 and 200 μg anti-CTLA-4 9D9. In the saline control group, tumours grew rapidly in all 8 mice until the humane endpoint was reached, with no mice surviving to day 30 after tumour cell implantation. In the 9D9 anti-CTLA-4 antibody treated group, tumour growth was slower relative to control, and 2 of 8 mice remained tumour free until the study endpoint. In the STIM003_574 treated group, tumour growth was also slower relative to control, and 1 of 8 mice remained tumour free until the study endpoint. A stronger tumour growth delay and anti-tumour effect was observed in the mice who received both STIM003_574 and anti-CTLA-4. Tumour growth was suppressed in all 8 mice, and 5 of 8 mice had no detectable tumour at the end of the study. FIG. 34 . Survival was extended, and overall survival was higher, in the combination therapy group compared with the group that received anti-CTLA-4 or STIM003_574 alone. FIG. 35 .

Example 18 Discovery and Characterisation of PD-L1 Binding Fcab

The anti-human PD-L1 Fcab included in the mAb2 molecules described in the preceding Examples was originally sourced through selection and screening of naïve Fcab phage libraries. Naïve Fcabs were further optimised for affinity and functionality in three rounds of affinity maturation. Binding affinities, cell binding, SEC-HPLC and activity in T cell activation assays, were the main assays used for screening.

Naïve selections on human PD-L1 led to the isolation of FS17-33, which binds specifically to cell-surface human PD-L1 and has blocking activity towards both PD-L1/PD-1 and PD-L1/CD80. This clone was subjected to three rounds of affinity maturation. Whereas the first round moderately increased affinity and blocking activity, the second round resulted in clones with high affinity and functional activity due to the engagement of the CD loop. In the third mutagenesis round a sequence liability was removed. A resulting clone FS17-33-289 was fully characterised and shown to bind human PD-L1 with low nanomolar affinities. This molecule was found to be cross-reactive for binding PD-L1 from cynomolgus monkey, but not from mouse. It showed activity in three different T cell activation assays that matched benchmark mAbs. Biophysical characterisation indicated that the lead Fcab was stable.

In summary, the naïve selections and subsequent affinity maturations for the FS17 project identified an anti-human PD-L1 Fcab that met all criteria in terms of affinity, functionality and biophysical characteristics, and was considered to represent an active and stable protein likely to have success in the clinic.

Naïve Phage Selections

unique clones were identified and subcloned into a Pichia pastoris expression vector (pPICzα, Invitrogen) for soluble expression. Soluble Fcab proteins were then analysed in an ELISA-based Receptor Binding Assay (RBA) to assess their ability to block the PD-L1:PD-1 interaction. In these assays, the ability of Fcabs to inhibit the binding of biotinylated hPD-L1 to immobilised hPD-1 or hCD80 was tested. Of the 30 clones tested, three clones, FS17-19, 26 and 33, showed potent PD-L1:PD-1 blocking activity.

FS17-33 showed potent PD-L1:PD-1 as well as PD-L1:CD80 blocking activity in ELISA-based RBAs. The activity of FS17-33 was about 15-20 fold off benchmark. The activity of FS17-33 to inhibit PD-L1/PD-1 interaction was also confirmed in a cell-based RBA

To test the binding specificity, FS17-33 binding was analysed at 1 μM on a panel of HEK293 cells overexpressing immune checkpoint regulators. FS17-33 bound specifically to cell-surface human PD-L1 and not to human PD-1, human CD80 or mouse PD-L1 and mouse CD80.

Affinity Maturation 1—Parsimonious Mutagenesis of Binding Loops

To increase the affinity of FS17-33, a parsimonious randomisation strategy was employed. In this strategy one single amino acid residue is randomised at a time, creating mini-libraries that can be scanned for improved affinity. The libraries were made with low-redundancy NNK codons designed to represent all possible AA in the position of interest. As FS17-33 contains five mutations in the AB and seven in EF loop compared to wild type Fc, 12 libraries were generated and expressed in HEK293 in a 96-well format. Supernatants were analysed for Fcab expression levels as well as blocking activity in PD-L1: PD-1 RBAs. The activity was compared to a FS17-33 dose-response curve used as a threshold for selection of improved mutants. Certain positions did not produce any improved blocking Fcabs indicating that the amino acids in these positions are crucial for the binding specificity. On the contrary, substitutions at other positions produced a number of improved Fcabs with sequence diversity.

27 improved Fcabs were re-expressed and re-analysed as purified protein. The top four AB mutants were subsequently shuffled with the top three EF mutants. These clones were ranked based on activity in cell-based RBA, cell binding, SEC-HPLC profile and activity in the T cell activation assay.

The blocking activity of the shuffled FS17-33 clones was assessed in cell-based RBAs using HEK293 overexpressing human PD-L1 and biotinylated hPD-1-Fc. Several FS17-33 derived mutants showed improved blocking activity compared to parental clone, albeit not matching benchmark mAb.

To test the binding to cell-surface human and cyno PD-L1, the top clones from replicate RBA assays were tested for binding to HEK293 overexpressing human or cyno PD-L1. All Fcabs showed dose-dependent binding to hPD-L1 with EC50 values around 5-10 nM. Binding to cell-bound cyno PD-L1 was of lower affinity (EC50 values around 100-200 nM). The affinity of Benchmark S1 was similar for both human and cyno PD-L1 indicating that HEK293: PD-1 and HEK293: cyno PD-L1 have similar levels of overexpression.

The functional activity of the FS17-33 derived clones was tested in a T cell activation assay. In this assay, T cells expressing PD-1 protein were used as effector cells and antigen presenting cells (APC) expressing human PD-L1 were used as target cells. The T cells were stimulated with the APCs in the presence or absence of FS17-33 derived Fcabs. After 24 hr incubation, the T cell reaction supernatants were harvested and assessed for IL-2 secretion by ELISA. The functional activity of the FS17-33 Fcabs was very weak compared to one of the benchmark mAb (EC50 of benchmarks are around 0.3 nM). The Fcabs were therefore screened at concentrations up to 2 μM to allow selection of the best clones.

Binding and activity data revealed a pool of Fcabs with similar activity. SEC-HPLC analysis was performed to analyse the percentage of monomer. FS17-33 derived Fcabs showed a 100% monomeric profile.

In conclusion the parsimonious mutagenesis strategy of parental FS17-33 Fcab has led to the identification of multiple clones with increased PD-L1: PD-1 blocking activity. The anti-human PD-L1 binding FS17-33 derived Fcabs are cross-reactive to cynoPD-L1 albeit with lower affinity. The activity of the FS17-33 lineage in the T cell activation assay was very weak compared to benchmark mAbs. This data seems inconsistent with the blocking data but could be explained by blocking assay parameters such as assay window and high levels of human PD-L1 on HEK293: PD-L1 causing avidity. More substantial randomisation of the binding loops is required to accomplish higher gains in affinity and functional activity. FS17-33-37 was selected as the parent for these affinity maturations.

Affinity Maturation 2—Hard Randomisation of Binding Loops

FS17-33-37 is a human-specific PD-L1 binding Fcab that is able to block the interaction between PD-L1:PD-1 and PD-L1:CD80 in vitro. The activity in T cell activation assay is weak and affinity maturation is therefore required. To this end the AB, CD and EF loops of FS17-33-37 were randomised and selections performed using the yeast platform. Enrichment for binders was observed during the selections and screening in the display format has identified anti-human FS17-33-37 derived Fcabs with superior binding to PD-L1. Sequence diversity was limited in the AB and EF loops while the CD loop showed high diversity with an emerging lineage. These clones were subcloned into the pTT5 mammalian expression vector for characterisation of the soluble protein. To screen the clones from the three selections, the supernatants from HEK293 expressed with Fcabs were flowed over a Biacore chip coated with hPD-L1-Fc. The off-rates were then compared to the parental clone. EF mutant clones showed little to no improvement and only a few clones from the AB selections showed improvement in the off-rate. However, all of the clones from the CD selections showed significant improvement in the off-rate. The best clones were then tested in the functional assay to correlate the improvement in off-rate with activity.

The best clone tested in the T-cell activation assay showed good improvement in activity compared to the parental clone, but was still considerably less active than benchmark mAbs. For this reason the clones with mutations in a single loop were deprioritized and it was decided to make a new shuffle library from the outputs of all three libraries.

The shuffled library was generated as described in the methods and to increase the diversity an error prone mutagenesis across the entire CH3 was added to the shuffle library. To identify the strongest binders and to minimise avidity, selections were performed with hPD-L1-His antigen biotinylated in-house. Since the biotinylation randomly occurs on lysines throughout the protein that could interfere with binding of the Fcabs, selections were also performed in parallel with hPD-L1-Fc-Avi antigen.

In each round the concentration was dropped and in the final round a second staining procedure was performed in which the antigen is placed in competition with non-biotinylated antigen to select clones with the best off-rates. An anti-CH2 antibody was included in all of the selections with the hPD-L1-His antigen as a marker for expression and proper folding of the Fcab. Since the hPD-L1-Fc antigen will also bind this antibody, it cannot be included as a co-stain during selections. In this case expression is marked by the anti-express antibody (Invitrogen) and an additional round is included to sort the clones with high anti-CH2 binding with the aim to remove improperly folded Fcabs. Both selections showed strong enrichment for binding above a parental clone selected from the individual loop selections.

Sequence analysis of the outputs from each round showed that the AB loop had converged to a single sequence not previously identified and the EF sequence was primarily parental. The CD loop showed good diversity with a strong consensus emerging. All of the outputs were then sub cloned into the pTT5 mammalian expression vector at the 10 mL scale. For the non-competition outputs, clones were first expressed at 1 mL to screen based on Biacore off-rate and single point T cell activation assay data. Positives were re-expressed at the 10 mL scale. In total 52 clones were expressed at the 10 mL expression scale and screened based on biophysical properties and full titration in the T cell activation assay. This was performed in two batches, the first batch contained clones that were from the competition outputs and did not need a primary screen at 1 mL. From this batch, the best clones were 10 to 30-fold off the benchmark mAbs in functional activity and exhibited favourable biophysical properties.

The hard randomisation of the loops was successful in improving the functional activity of clone FS17-33-37. Most of the gains in affinity came from mutations in the CD and AB loops. The best clones from this campaign exhibit functional activity that matches benchmark mAbs in the T-cell activation assay. These clones contain a methionine in the EF loop which was present in the parental clone FS17-33-37.

Affinity Maturation 3—Parsimonious Mutagenesis of Methionine

To remove the methionine from the EF loop of the lead Fcabs, a parsimonious mutagenesis maturation strategy was initiated. At the methionine amino acid position in the EF loop, the sequence identity was randomised and all of the amino acid substitutions that did not introduce sequence liabilities were tested for binding, SEC profile and functional activity.

Mutations M419L was able to remove the methionine from the EF loop without any loss in activity. This mutation generated clone FS17-33-289.

Initial Characterisation of PD-L1 Fcab

Fcab binding affinity towards monomeric human and cyno PD-L1-His was determined using Biacore by immobilising the Fcab or mAb using Protein A capture and measuring the binding to PD-L1. The Fcabs bound to monomeric human PD-L1 with low nanomolar affinity and binding to cyno PD-L1 was within 2-fold of the binding to human PD-L1.

To determine whether the Fcabs specifically bind to cell-surface PD-L1, Fcab binding to HEK293 control cells or HEK293 cells overexpressing either human or cyno PD-L1 was assessed. Consistent with the Biacore binding, FS17-33-289 showed specific binding to cell-surface human and cyno PD-L1.

The ability of the anti-human PD-L1 binding Fcab to block the PD-1/PD-L1 as well as the CD80/PD-L1 interaction was tested in cell-based blocking assays. The data showed that FS17-33-289 efficiently blocked both the PD-1/PD-L1 and the CD80/PD-L1 interaction in vitro.

Inhibition of PD-1/PD-L1 and CD80/PD-L1 interaction relieves the co-inhibitory signals after T cell activation. The functional activity of the anti-human PD-L1 binding Fcabs was first tested in the previously described T cell activation assay using a T cell hybridoma cell line and APCs overexpressing human PD-L1. FS17-33-289 was active in this assay, and activity was further confirmed in a Staphylococcal Enterotoxin B (SEB) assay and a Mixed Leukocyte Reaction (MLR) assay using human PBMCs.

The ability of the anti-human PD-L1 binding Fcabs to bind to Protein A was determined using the Octet by assessing the binding of purified FS17-33-289 and wild type Fc protein to Protein A tips. Fcab showed similar binding to Protein A as wild type Fc.

The thermal stability of the anti-human PD-L1 Fcab was analysed by differential scanning calorimetry (DCS). A DSC thermogram of wild type Fc was used to represent the unfolding of the CH2 domain at 71° C. and CH3 domain at 83° C. domain, respectively. DSC analysis of the anti-human PD-L1 binding Fcab gave rise to a single melting transition, as is typically seen in Fcabs upon modification of the AB, CD and EF loops. The melting temperature differential of the Fcab compared to wild type Fc CH2 domain was within 5° C. and compared to the wild type Fc CH3 domain it was about 16° C.

In order to test Fcab stability, FS17-33-289 protein was incubated for 7 days in 90% serum at 37° C. Control samples were either kept at 4° C. or incubated in serum before adding to the assay (‘0 day serum’). The activity was subsequently analysed in the T cell activation assay. Activity of the Fcabs incubated in serum for 7 days was equivalent to the control samples as well as to benchmark.

Materials & Methods Naïve Phage Selections

Four Fcab phage libraries, PP1, PP2 PP4 and 5, were used for human naïve selections. For each library, three rounds of selections were performed using a combination of non-biotinylated and biotinylated hPD-L1-Fc antigens as wells as HEK293 cells overexpressing human PD-L1. For the recombinant antigen selections the beads used were alternated between streptavidin and neutravidin to avoid selecting phage that bound to either of those proteins. To deplete potential Fc binders from the input population a 20-fold excess of non-biotinylated recombinant human Fc protein was used. For the cell selections, control HEK293 cells were used for the deselection of non-specific cell binders.

FCAB Cloning and Expression

Phage or yeast pellets of individual clones were lysed and the CH3 region amplified by PCR before cloning into the pTT5 expression vector (National Research Council of Canada, NRC) engineered with an effector-less CH2 domain (LALA mutation). Expi293F™ cells from Invitrogen™ were grown according to the manufacturer's specifications and used for transfections at a cell density of 2×10⁶ cells/mL. Transfections were performed using Expifectamine™ combined with the purified DNA in Optimem™ and added to 1 mL cell cultures for screening or 20 mL for protein characterisation. The cells were cultured for 5 days, followed by centrifugation to separate the cells from the supernatant. For screening assays, cell culture supernatants were collected and Fcab expression was titered by binding to protein-A tips using the Octet. Fcab concentrations in the supernatant ranged from 30 to 700 μg/ml. Aliquots of supernatant were then buffer exchanged using a PD MultiTrap G-25 in PBS to measure antigen binding or in DMEM media for further characterisation. During the naïve selection campaigns Fcabs were initially expressed using the Pichia pastoris. To this end, the Fcabs were cloned into pPICzα and expressed exactly as described by the manufacturer (Invitrogen, Pichia expression kit).

Affinity Maturation Library Generation

For affinity maturation, Fcabs from prior campaigns were selected based on their activity in the functional DO11 assay, SEC-HPLC profiles (>80% monomeric) and sequence diversity. To reduce the presence of parental clones in the libraries, wild type Fc sequences were restored in the AB and EF loops before mutagenesis of the respective loop. Full diversification of the AB, CD or EF loops was achieved by mutagenesis PCR. Library sizes covered the theoretical size 2 to 500-fold, except for the EF libraries in which the diversity is not covered. Sequence diversity was verified for all the libraries.

Loop Shuffle and Error Prone Yeast Mini Library Generation

The outputs from phage that showed the best off-rates and highest percentage of binders were used to create a shuffled library with error prone mutagenesis randomisation. The individual AB, CD, and EF loops were isolated, amplified via PCR and assembled randomly. Shuffled DNA fragments were subsequently amplified using the Gene Morph II random mutagenesis kit (Agilent) aiming for one to two mutations per CH3 domain. The resulting DNA was transformed into the yeast pYD1 vector (Invitrogen) containing the CH2 domain to produce an Fcab mini-library for yeast selections.

Yeast Affinity Maturation Selections

Yeast affinity maturation libraries were subjected to two to four rounds of selections using biotinylated hPD-L1-His protein. In each round of selection, the library as well as the parental clone were incubated with biotinylated antigen and binders were separated from non-binders by fluorescent cell sorting (FACS). Clones with improved binding were selected by gating higher fluorescent intensity cells those of the parental clone. Antigen concentrations were dropped in consecutive rounds to increase the stringency of the selection. In the final selection round an off-rate step was included which involved incubating the sample for several hours at room temperature in the presence of excess non-biotinylated antigen.

Subcloning from Yeast

Individual clones or outputs from selections were subcloned into the pTT5 mammalian expression vector (National Research Council of Canada, NRC) to produce soluble protein for screening and characterisation. DNA encoding the Fcab CH3 domain was amplified by PCR directly from phage glycerol stocks or from yeast that had been lysed using lyticase. Amplified DNA was subsequently digested and cloned into the pTT5 vector containing the IgG1 CH2 domain. For loop shuffling, the AB, CD and EF loops of selection outputs were combined by using a PCR to amplify either loop, followed by an assembly reaction to create the desired loop combinations. Shuffled DNA fragments were subsequently amplified using the Gene Morph II random mutagenesis kit (Agilent) aiming for one to two mutations per CH3 domain. The resulting DNA was transformed into the yeast pYD1 vector (Invitrogen) containing the CH2 domain to produce an Fcab mini-library for yeast selections.

Parsimonious Mutagenesis

Single point mutations were introduced in the AB and the EF loops of FS17-33-116 using the Quick-Change site directed mutagenesis kit (Agilent) and NNK randomisation primers. Mutations likely to introduce sequence liabilities were excluded. After transformation into E. coli, the DNA sequences of each mutant were verified and DNA was extracted for transient transfection into HEK293 cells.

Off-Rate Screening

HEK293 supernatants or purified proteins were screened for improvements in the off-rate using the Biacore 2000. Biotinylated hPD-L1-His was immobilised on streptavidin chips at approximately 216-228 RU for an expected Rmax of 100. Regeneration was achieved with one 30 sec. injection of 10 mM Glycine pH 2.5 followed by one 30 sec. injection of 10 mM NaOH. Purified proteins or HEK293 supernatants containing soluble Fcabs were injected over the chip at a flow rate of 30-50 μl/min for three minutes followed by a dissociation step at the same flow rate. Parental clones were included in the screening assay for comparison.

FCAB Purification

Fcabs expressed in 20 ml cultures were purified using MabSelect SuRe™ resin under endotoxin-free conditions. Eluted Fcab was buffer exchanged into PBS using and concentrated to at least 1 mg/ml using Amicon Ultra centrifugal units. Endotoxin levels were determined using Limulus Amebocyte Lysate Endochrome assay as described by the manufacturer (Endosafe kit, Charles River). Proteins with endotoxin levels <0.15 EU/mg were considered acceptable.

T Cell Activation Assay

Mouse antigen (Ovalbulmin)-specific T cells expressing murine PD-1 protein were used as effector cells and mouse antigen presenting cells (APC) expressing hPD-L1 were used as target cells. The T cells were stimulated with the APCs in the presence or absence of anti-hPD-L1 Fcab or anti-hPD-L1 benchmark antibodies at various concentrations and ovalbumin peptide. In the assay reaction, anti-hPD-L1 Fcabs were added either as purified protein or as HEK293 supernatant. After 24 hr incubation, the T cell reaction supernatants were harvested and assessed for IL-2 secretion by ELISA.

Mixed Lymphocyte Reaction (MLR) Assay

The MLR is an allogeneic reaction. Expanded CD4+ T cells from donor A are used as effector cells while immature dendritic cells (iDCs) from donor B are the APCs. The donor mismatch causes an immune response which results in activation of the TCR and release of IFNγ. After several days, increasing expression of hPD-L1 on the APCs with hPD-1 on the T cells inhibits IFNγ production. The addition of anti-human PD-L1 specific Fcabs blocks the interaction of hPD-L1 with hPD-1, releasing hPD-1 inhibition, and allowing IFNγ production. In this assay, Fcabs (and controls) are incubated with 1×10⁵ CD4+ T cells and 1×10⁴ iDCs per well for 5 days. The supernatants are then harvested and assessed for hIFNγ secretion by ELISA.

Staphylococcal Enterotoxin (SEB) Assay

In this assay expanded CD4+ T cells and iDCs from the same donor are mixed. SEB acts as a superantigen, being presented by MHC class II on the iDCs to the TCR on the T cells. As in the MLR, hPD-L1 on the iDCs interacts with PD-1 on the T cells, blocking IFNγ production. With the addition of anti-human PD-L1 Fcabs the block is released and IFNγ produced. Fcabs (and controls) are incubated with 1×10⁵ CD4+ T cells and 1×10⁴ iDCs per well with 0.1 ng/ml SEB for 4 days. The supernatants are then harvested and assessed for hIFNγ secretion by ELISA.

SEC-HPLC

SEC-HPLC experiments were performed on an Agilent 1100 series HPLC with a Zorbax GF-250 column (Agilent). The flow rate for these experiments was 1 ml/min and the mobile phase was 200 mM sodium phosphate, 400 mM NaCl, 15% isopropanol, pH 6.8. Sample concentrations were 0.5-1 mg/ml in PBS buffer.

Affinity Analysis

The affinity of the Fcabs was measured using the Biacore T200. Fcabs were captured using a Protein A immobilised chip. Human PD-L1-His binding was assessed using flow rates of 75 μl/min. Regeneration was achieved with one 30 sec. injection of 10 mM glycine pH 2.0. Curves were fit using the 1:1 model in the T200 evaluation software with Rmax set to local and RI set to 0.

Cell Binding

Cell binding assays were performed by incubating Fcabs or mAb proteins with either control HEK293 or HEK293 overexpressing hPD-L1 or cyno PD-L1 cells for 1 hour at 4° C. Fcab/mAb binding was subsequently detected using an anti-human Fc 488 pre-labeled detection antibody. Fluorescent signal intensities were measured using the FACS Canto.

Protein A Binding

Fcabs were serially diluted 1:2 in kinetics buffer starting from 2 μM in 1× kinetics buffer and assayed for binding to Protein A tips in the Octet (Forte Bio). All curves were subtracted from a reference well and grouped based on the Fcab being assayed.

Cell-Based Blocking Assays

The blocking activity of the Fcabs on both hPD-1/hPD-L1 and hPD-L1/hCD80 interactions was tested in cell-based Receptor Binding Assays (RBA). In brief, biotinylated hPD-L1-Fc-Avi at 1 μg/ml was incubated for 1 hour with titrating concentrations of Fcabs ranging from 400 nM to 3 pM. The mix was incubated for another hour with HEK293 cells overexpressing either hPD-1 or hCD80. The level of bound biotinylated hPD-L1-Fc-Avi on the cells was detected using streptavidin 647 and fluorescence levels were measured using the FACS Canto. Each point was performed in duplicate and experiments were performed twice. For the experiment performed with HEK293:hCD80 cells, the concentration of biotinylated hPD-L1-Fc-Avi used was 2 μg/ml.

DSC

Thermal stability measurements were performed using a Microcal VP-capillary differential scanning calorimeter (DSC). Samples were measured in PBS buffer (Lonza) at 0.2 mg/ml. The scan rate was set at 60° C./hr and data collected between 30° C. and 95° C. Data analysis was performed with Origin 7.0 software.

Sequences

TABLE S1 SEQ ID NOS: 1-342 SEQ   ID NO: Name Description Sequence 1 Human NCBI number: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEK PD-L1 NP_054862.1 QLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL (ECD highlighted SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKIN in BOLD, QRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNS cytoplasmic domain KREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELP underlined) LAHPPNERT HLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQD TNSKKQSDTHLEET 2 Cyno PD- NCBI number: MGWSCIILFLVATATGVHSMFTVTVPKDLYVVEYGSNMTIECKFPV L1 XP_014973154.1 EKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKD (ECD highlighted QLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNK in BOLD) INQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTT NSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPE LPLALPPNERT 3 Human Human PD-L1 ECD MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEK His His tag QLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL PD-L1 with C-terminal SIGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKIN QRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNS KREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELP LAHPPNERT HHHHHH 4 Human Human PD-L1 ECD MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEK PD-L1 Fc with C-term Fc QLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL fusion (in bold) SIGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKIN QRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNS KREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELP LAHPPNERT IEGREPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 5 Cyno PD- Cynomolgus PD-L1 MGWSCIILFLVATATGVHSMFTVTVPKDLYVVEYGSNMTIECKFPV L1 FLAG ECD with N-term EKQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKD QLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNK INQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTT NSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPE LPLALPPNERT DYKDDDDK 6 Human Human PD-1 full MGWSCIILFLVATATGVHSLDSPDRPWNPPTFSPALLVVTEGDNAT PD-1 Fc FLAG tag FTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV length sequence TQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELR derived from cDNA VTERRAEVPTAHPSPSPRPAGQ KLENLYFQGIEGRMDEPKSCDKTH as human Fc fusion TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 7 84G09- Amino acid GFTFDDYA CDRH1 sequence of CDRH1 (IMGT) of 84G09 using IMGT 8 84G09 Amino acid ISWKSNII CDRH2 sequence of CDRH2 (IMGT) of 84G09 using IMGT 9 84G09 Amino acid ARDITGSGSYGWFDP CDRH3 sequence of CDRH3 (IMGT) of 84G09 using IMGT 10 84G09 Amino acid DYAMH CDRH1 sequence of CDRH1 (Kabat) of 84G09 using Kabat 11 84G09- Amino acid GISWKSNIIGYADSVKG CDRH2 sequence of CDRH2 (Kabat) of 84G09 using Kabat 12 84G09- Amino acid DITGSGSYGWFDP CDRH3 sequence of CDRH3 (Kabat) of 84G09 using Kabat 13 84G09- Amino acid EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLE Heavy sequence of V_(H) of WVSGISWKSNIIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA chain 84G09 (mutations LYYCARDITGSGSYGWFDPWGQGTLVTVSS variable from germline are region shown in bold letters) 14 84G09- Nucleic acid CAaGAAAAAGCTTGCCGCCACCATGGAGTTTGGGCTGAGCTGGATT Heavy sequence of V_(H) of TTCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTACAATTGG chain 84G09 TGGAGTCCGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACT variable CTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCAC region TGGGTCCGACAAACTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA TAAGTTGGAAGAGTAATATCATAGGCTATGCGGACTCTGTGAAGGG CCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTG CAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTATTGTG CAAGAGATATAACGGGTTCGGGGAGTTATGGCTGGTTCGACCCCTG GGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAAAACGACACCC CCATCTGTCTATCCACTGGCCCCTGAATCTGCTAAAACTCAGCCTC CG 15 84G09- Amino acid EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLE full sequence of 84G09 WVSGISWKSNIIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTA heavy heavy chain LYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPLAPCSR chain (mutations from STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL sequence germline are shown YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP in bold letters) CPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 16 84G09 Nucleic acid GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCA full sequence of 84G09 GATCCCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCGACGA heavy heavy chain CTACGCTATGCACTGGGTGCGACAGACCCCTGGCAAGGGCCTGGAA chain TGGGTGTCCGGCATCTCCTGGAAGTCCAACATCATCGGCTACGCCG sequence ACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACGCCAAGAA CTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCC CTGTACTACTGCGCCAGAGACATCACCGGCTCCGGCTCCTACGGAT GGTTCGATCCTTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGC CAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAG TCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACT ACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGAC CAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTG TACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCA CCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAA GGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACC TGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGT TCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGAC CCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCT GAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACG CCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGT GGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAA GAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCG AAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGT GTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTG TCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCG TGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCAC CCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAG CTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCT GCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTC CCTGTCCCTGAGCCCCGGCAAG 17 84G09- Amino acid QSISSY CDRL1 sequence of CDRL1 (IMGT) of 84G09 using IMGT 18 84G09- Amino acid VAS CDRL2 sequence of CDRL2 (IMGT) of 84G09 using IMGT 19 84G09- Amino acid QQSYSNPIT CDRL3 sequence of CDRL3 (IMGT) of 84G09 using IMGT 20 84G09- Amino acid RASQSISSYLN CDRL1 sequence of CDRL1 (Kabat) of 84G09 using Kabat 21 84G09- Amino acid VASSLQS CDRL2 sequence of CDRL2 (Kabat) of 84G09 using Kabat 22 84G09 Amino acid QQSYSNPIT CDRL3 sequence of CDRL3 (Kabat) of 84G09 using Kabat 23 84G09- Amino acid DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKP Light sequence of V_(L) of LIYVASSLQSGVPSSFSGSGSGTDFTLTISSLQPEDFATYYCQQSY chain 84G09 SNPITFGQGTRLEIK variable region 24 84G09- Nucleic acid GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG Light sequence of V_(L) of GAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAG chain 84G09 CTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCCC variable CTGATCTATGTTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGTT region TCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG TCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTAC AGTAATCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAA 25 84G09- Amino acid DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKP full sequence of 84G09 LIYVASSLQSGVPSSFSGSGSGTDFTLTISSLQPEDFATYYCQQSY light light chain SNPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 26 84G09- Nucleic acid GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG  full sequence of 84G09 GAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAG light light chain CTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCCC  CTGATCTATGTTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGTT  TCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG  TCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTAC  AGTAATCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC  GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA  GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC  TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC  TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA  GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC  GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG  GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 27 1D05- Amino acid GFTFDDYA CDRH1 sequence of CDRH1 (IMGT) of 1D05 using IMGT chain 28 1D05- Amino acid ISWIRTGI CDRH2 sequence of CDRH2 (IMGT) of 1D05 using IMGT sequence 29 1D05- Amino acid AKDMKGSGTYGGWFDT CDRH3 sequence of CDRH3 (IMGT) of 1D05 using IMGT 30 1D05- Amino acid DYAMH CDRH1 sequence of CDRH1 (Kabat) of 1D05 using Kabat 31 1D05- Amino acid GISWIRTGIGYADSVKG CDRH2 sequence of CDRH2 (Kabat) of 1D05 using Kabat 32 1D05- Amino acid DMKGSGTYGGWFDT CDRH3 sequence of CDRH3 (Kabat) of 1D05 using Kabat 33 1D05- Amino acid EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLE Heavy sequence of V_(H) of WVSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTA chain 1D05 (mutations LYYCAKDMKGSGTYGGWFDTWGQGTLVTVSS variable from germline are region shown in bold letters) 34 1D05- Nucleic acid AAGCTTGCCGCCACCATGGAGTTTGGGCTGAGCTGGATTTTCCTTT Heavy sequence of V_(H) of TGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTC chain 1D05 TGGGGGAGGCTTGGTGCAGCCTGGCAGGTCCCTGAGACTCTCCTGT variable GCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCC region GGCAAGTTCCAGGGAAGGGCCTGGAATGGGTCTCAGGCATTAGTTG GATTCGTACTGGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTC ACCATTTTCAGAGACAACGCCAAGAATTCCCTGTATCTGCAAATGA ACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGA TATGAAGGGTTCGGGGACTTATGGGGGGTGGTTCGACACCTGGGGC CAGGGAACCCTGGTCACCGTCTCCTCAGCCAAAACAACAGCCCCAT CGGTCTATCCACTGGCCCCTGC 35 1D05- Amino acid EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLE full sequence of 1D05 WVSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTA heavy heavy chain LYYCAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCS chain RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 36 1D05- Nucleic acid GAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCA full sequence of 1D05 GATCCCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCGACGA heavy heavy chain CTACGCTATGCACTGGGTGCGACAGGTGCCAGGCAAGGGCCTGGAA chain TGGGTGTCCGGCATCTCTTGGATCCGGACCGGCATCGGCTACGCCG sequence ACTCTGTGAAGGGCCGGTTCACCATCTTCCGGGACAACGCCAAGAA CTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCC CTGTACTACTGCGCCAAGGACATGAAGGGCTCCGGCACCTACGGCG GATGGTTCGATACTTGGGGCCAGGGCACCCTCGTGACCGTGTCCTC TGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGC AAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGG ACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCT GACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGC CTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGG GCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACAC CAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCAC ACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCG TGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCG GACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGAC CCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACA ACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCG GGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCA TCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCA GGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAG GTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCG CCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGAC CACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGC AAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCT CCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA GTCCCTGTCCCTGAGCCCCGGCAAG 37 1D05- Amino acid QSISSY of 1D05 using IMGT CDRL1 sequence of CDRL1 (IMGT) 38 1D05- Amino acid VAS CDRL2 sequence of CDRL2 (IMGT) of 1D05 using IMGT 39 1D05- Amino acid QQSYSTPIT CDRL3 sequence of CDRL3 (IMGT) of 1D05 using IMGT 40 1D05- Amino acid RASQSISSYLN CDRL1 sequence of CDRL1 (Kabat) of 1D05 using Kabat 41 1D05- Amino acid VASSLQS CDRL2 sequence of CDRL2 (Kabat) of 1D05 using Kabat 42 1D05- Amino acid QQSYSTPIT CDRL3 sequence of CDRL3 (Kabat) of 1D05 using Kabat 43 1D05- Amino acid DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL Light sequence of V_(L) of LIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY chain 1D05 (mutations STPITFGQGTRLEIK variable from germline are region shown in bold letters) 44 1D05- Nucleic acid AAAGCTTGCCGCCACCATGAGGCTCCCTGCTCAGCTTCTGGGGCTC Light sequence of V_(L) of CTGCTACTCTGGCTCCGAGGTGCCAGATGTGACATCCAGATGACCC chain 1D05 AGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCAT variable CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTAT region CAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGTTGCAT CCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC TGGGACAGATTTCACTCTCACTATCAGCAGTCTGCAACCTGAAGAT TTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGATCACCT TCGGCCAAGGGACACGTCTGGAGATCAAACGTACGGATGCTGCACC AACT 45 1D05- Amino acid DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL full sequence of 1D05 LIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY light light chain STPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 46 1D05- Nucleic acid GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCTTCCGTGG full sequence of 1D05 GCGACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCTC light light chain CTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTG chain CTGATCTACGTGGCCAGCTCTCTGCAGTCCGGCGTGCCCTCTAGAT sequence TCTCCGGCTCTGGCTCTGGCACCGACTTTACCCTGACCATCAGCTC CCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTCCTAC TCCACCCCTATCACCTTCGGCCAGGGCACCCGGCTGGAAATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 47 Mutated Amino acid EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ A PGKGLE 1D05- sequence of 1D05 WVSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTA HC heavy chain with V LYYCAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCS mutant 1 to A back-mutation RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG in framework LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP region to germline PCPAPE LAGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV highlighted with QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY IgG1 disabled KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL (LAGA) constant TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT region VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 48 Mutated Amino acid EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLE 1D05- sequence of 1D05 WVSGISWIRTGIGYADSVKGRFTI S RDNAKNSLYLQMNSLRAEDTA HC heavy chain with F LYYCAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCS mutant 2 to S back-mutation RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG in framework LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP region to germline PCPAPE LAGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV highlighted with QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY IgG1 disabled KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL (LAGA) constant TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT region VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 49 Mutated Amino acid EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLE 1D05- sequence of 1D05 WVSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTA HC heavy chain with LYYCAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCS mutant 3 ELLG to -PVA back- RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG mutation in LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP constant region to PCPAP- germline PVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV highlighted DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK 50 Mutated Amino acid DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL 1D05- sequence of 1D05 LIY A ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY LC kappa light chain STPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN mutant 1 with V to A back- FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA mutation in CDRL2 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC to germline highlighted 51 Mutated Amino acid DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL 1D05- sequence of 1D05 F IYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY LC kappa light chain STPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN mutant 2 with L to F back- FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA mutation in DYEKHKVYACEVTHQGLSSPVTKSFNRGEC framework to germline highlighted 52 411B08- Amino acid GFTFSSYW CDRH1 sequence of (IMGT) CDRH1 of 411B08 Using IMGT 53 411B08- Amino acid IKEDGSEK CDRH2 sequence (IMGT) of CDRH2 of 411B08 using IMGT 54 411B08- Amino acid ARNRLYSDFLDN CDRH3 sequence (IMGT) of CDRH3 of 411B08 Using IMGT 55 411B08- Amino acid SYWMS CDRH1 sequence of CDRH1 (Kabat) of 411B08 using Kabat 56 411B08- Amino acid NIKEDGSEKYYVDSVKG CDRH2 sequence of CDRH2 (Kabat) of 411B08 using Kabat 57 411B08- Amino acid NRLYSDFLDN CDRH3 sequence of CDRH3 (Kabat) of 411B08 using Kabat 58 411B08- Amino acid EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE Heavy sequence of V_(H) of WVANIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTS chain 411B08 VYYCARNRLYSDFLDNWGQGTLVTVSS variable region 59 411B08 Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG Heavy sequence of V_(H) of GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAG chain 411B08 CTATTGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG variable TGGGTGGCCAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCG region ACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCT GTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACA ACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG 60 411B08- Amino acid EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE full sequence of WVANIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTS heavy 411B08 VYYCARNRLYSDFLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTS chain heavy chain GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL sequence SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 61 411B08 Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG full sequence of GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAG heavy 411B08 CTATTGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG chain heavy chain TGGGTGGCCAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCG sequence ACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCT GTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACA ACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAA GGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCT GGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCG AGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGT GCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTG TCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCT ACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA GAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCT TGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCC CCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGT GACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCA AGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGT GCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAG TGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCA TCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACT GCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACC TGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGG AGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTG GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGA TGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCT GAGCCCCGGCAAG 62 411B08- Amino acid QGVSSW CDRL1 sequence of CDRL1 (IMGT) of 411B08 using IMGT 63 411B08- Amino acid GAS CDRL2 sequence of CDRL2 (IMGT) of 411B08 using IMGT 64 411B08 Amino acid QQANSIPFT CDRL3 sequence of CDRL3 (IMGT) of 411B08 using IMGT 65 411B08- Amino acid RASQGVSSWLA CDRL1 sequence of CDRL1 (Kabat) of 411B08 using Kabat 66 411B08- Amino acid GASSLQS CDRL2 sequence of CDRL2 (Kabat) of 411B08 using Kabat 67 411B08- Amino acid QQANSIPFT CDRL3 sequence of CDRL3 (Kabat) of 411B08 using Kabat 68 411B08- Amino acid DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKL Light sequence of V_(L) of LIYGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQAN chain 411B08 SIPFTFGPGTKVDIK variable region 69 411B08- Nucleic acid GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCG Light sequence of V_(L) of GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAG chain 411B08 CTGGTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTC variable CTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGAT region TCAGCGGCAGTGGATCTGGGACAGAGTTCATTCTCACCATCAGCAG CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAAC AGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC 70 411B08- Amino acid DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKL full sequence of 411B08 LIYGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQAN light light chain SIPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 71 411B08 full Nucleic acid GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCG Light sequence of GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAG chain 411B08 CTGGTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTC sequence light chain CTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGAT TCAGCGGCAGTGGATCTGGGACAGAGTTCATTCTCACCATCAGCAG CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAAC AGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 72 411C04- Amino acid GFTFSSYW  CDRH1 (IMGT) sequence of CDRH1 of 411C04 using IMGT 73 411C04= Amino acid IKEDGSEK CDRH2 (IMGT) sequence of CDRH2 of 411C04 using IMGT 74 411C04- Amino acid ARVRLYSDFLDY CDRH3 (IMGT) sequence of CDRH3 of 411C04 using IMGT 75 411C04- Amino acid SYWMS CDRH1 (Kabat) sequence of CDRH1 of 411C04 using Kabat 76 411C04- Amino acid NIKEDGSEKYYVDSLKG CDRH2 (Kabat) sequence of CDRH2 of 411C04 using Kabat 77 411C04- Amino acid VRLYSDFLDY CDRH3 (Kabat) sequence of CDRH3 of 411C04 using Kabat 78 411C04- Amino acid EVQLVDSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE Heavy sequence of V_(H) Of WVANIKEDGSEKYYVDSLKGRFTISRDNAKNSLYLQMNSLRAEDTS chain 411C04 VYYCARVRLYSDFLDYWGQGTLVTVSS variable region 79 411C04- Nucleic acid GAGGTGCAGCTGGTGGACTCTGGGGGAGGCTTGGTCCAGCCTGGGG Heavy sequence of V_(H) of GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAG chain 411C04 CTATTGGATGAGTTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAG variable TGGGTGGCCAACATAAAAGAAGATGGAAGTGAGAAATACTATGTAG region ACTCTTTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA CTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCT GTGTATTACTGTGCGAGAGTTCGACTCTACAGTGACTTCCTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG 80 411C04- Amino acid EVQLVDSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE full sequence of 411C04 WVANIKEDGSEKYYVDSLKGRFTISRDNAKNSLYLQMNSLRAEDTS heavy heavy chain VYYCARVRLYSDFLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS chain GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL sequence SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 81 411C04- Nucleic GAGGTGCAGCTGGTGGACTCTGGGGGAGGCTTGGTCCAGCCTGGGG full acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAG heavy sequence CTATTGGATGAGTTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAG chain of TGGGTGGCCAACATAAAAGAAGATGGAAGTGAGAAATACTATGTAG sequence 411C04 ACTCTTTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA heavy CTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCT chain GTGTATTACTGTGCGAGAGTTCGACTCTACAGTGACTTCCTTGACT ACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAA GGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCT GGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCG AGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGT GCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTG TCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCT ACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA GAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCT TGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCC CCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGT GACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCA AGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGT GCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAG TGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCA TCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACT GCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACC TGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGG AGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTG GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGA TGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCT GAGCCCCGGCAAG 82 411C04- Amino QGVSSW CDRL1 acid (IMGT) sequence of CDRL1 of 411C04 using IMGT 83 411C04- Amino GAS CDRL2 acid (IMGT) sequence of CDRL2 of 411C04 using IMGT 84 411C04- Amino acid QQANSIPFT CDRL3 sequence of CDRL3 (IMGT) of 411C04 using IMGT 85 411C04 Amino acid RASQGVSSWLA CDRL1 sequence of CDRL1 (Kabat) of 411C04 using Kabat 86 411C04- Amino acid GASSLQS CDRL2 sequence of CDRL2 (Kabat) of 411C04 using Kabat 87 411C04- Amino acid QQANSIPFT CDRL3 sequence of CDRL3 (Kabat) of 411C04 using Kabat 88 411C04- Amino acid DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKL Light sequence of V_(L) of LIYGASSLQSGVPSRFSGSGSGTEFILSISSLQPEDFATYYCQQAN chain 411C04 SIPFTFGPGTKVDIK variable region 89 411C04- Nucleic acid GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCG Light sequence of V_(L) of GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAG chain 411C04 TTGGTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTC variable CTGATCTATGGTGCCTCCAGTTTGCAAAGTGGGGTCCCATCAAGAT region TCAGCGGCAGTGGATCTGGGACAGAGTTCATTCTCAGCATCAGCAG CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAAC AGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC 90 411C04- Amino acid DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKL full sequence of 411C04 LIYGASSLQSGVPSRFSGSGSGTEFILSISSLQPEDFATYYCQQAN light light chain SIPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 91 411C04- Nucleic GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCG full acid GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAG light sequence TTGGTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTC chain of CTGATCTATGGTGCCTCCAGTTTGCAAAGTGGGGTCCCATCAAGAT sequence 411C04 TCAGCGGCAGTGGATCTGGGACAGAGTTCATTCTCAGCATCAGCAG light CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAAC chain AGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 92 411D07- Amino GGSIISSDW CDRH1 acid (IMGT) sequence of CDRH1 of 411D07 using IMGT 93 411D07 Amino IFHSGRT CDRH2 acid (IMGT) sequence of CDRH2 of 411D07 using IMGT 94 411D07- Amino ARDGSGSY CDRH3 acid (IMGT) sequence of CDRH3 of 411D07 using IMGT 95 411D07- Amino SSDWWN CDRH1 acid (Kabat) sequence of CDRH1 of 411D07 using Kabat 96 411D07- Amino EIFHSGRTNYNPSLKS CDRH2 acid (Kabat) sequence of CDRH2 of 411D07 using Kabat 97 411D07- Amino DGSGSY CDRH3 acid (Kabat) sequence of CDRH3 of 411D07 using Kabat 98 411D07 Amino QVQLQESGPGLVKPSGTLSLTCIVSGGSIISSDWWNWVRQPPGKGL Heavy acid EWIGEIFHSGRTNYNPSLKSRVTISIDKSKNQFSLRLSSVTAADTA chain sequence VYYCARDGSGSYWGQGTLVTVSS variable of region V_(H) of 411D07 99 411D07- Nucleic CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG Heavy acid GGACCCTGTCCCTCACCTGCATTGTCTCTGGTGGCTCCATCATCAG chain sequence TAGTGACTGGTGGAATTGGGTCCGCCAGCCCCCAGGGAAGGGGCTG variable of GAGTGGATTGGAGAAATCTTTCATAGTGGGAGGACCAACTACAACC region V_(H) CGTCCCTCAAGAGTCGAGTCACCATATCAATAGACAAGTCCAAGAA of TCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCGGACACGGCC 411D07 GTGTATTACTGTGCGAGAGATGGTTCGGGGAGTTACTGGGGCCAGG GAACCCTGGTCACCGTCTCCTCAG 100 411D07- Amino QVQLQESGPGLVKPSGTLSLTCIVSGGSIISSDWWNWVRQPPGKGL full acid EWIGEIFHSGRTNYNPSLKSRVTISIDKSKNQFSLRLSSVTAADTA heavy sequence VYYCARDGSGSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA chain of ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV sequence 411D07 TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP heavy ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY chain VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 101 411D07- Nucleic CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG full acid GGACCCTGTCCCTCACCTGCATTGTCTCTGGTGGCTCCATCATCAG heavy sequence TAGTGACTGGTGGAATTGGGTCCGCCAGCCCCCAGGGAAGGGGCTG chain of GAGTGGATTGGAGAAATCTTTCATAGTGGGAGGACCAACTACAACC sequence 411D07 CGTCCCTCAAGAGTCGAGTCACCATATCAATAGACAAGTCCAAGAA heavy TCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCGGACACGGCC chain GTGTATTACTGTGCGAGAGATGGTTCGGGGAGTTACTGGGGCCAGG GAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGT GTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCC GCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCG TGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCC TGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTG ACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACG TGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACC CAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCT GAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCA AGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGT GGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGG AACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCT GCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC AACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCA AGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAG GGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAA GGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCC AGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGA CGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGG TGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCC TGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAA G 102 411D07- Amino QSVLYSSNNKNY CDRL1 acid (IMGT) sequence of CDRL1 of 411D07 using IMGT 103 411D07- Amino WAS CDRL2 acid (IMGT) sequence of CDRL2 of 411D07 using IMGT 104 411D07- Amino QQYYSNRS CDRL3 acid (IMGT) sequence of CDRL3 of 411D07 using IMGT 105 411D07- Amino KSSQSVLYSSNNKNYLA CDRL1 acid (Kabat) sequence of CDRL1 of 411D07 using Kabat 106 411D07- Amino WASTRES CDRL2 acid (Kabat) sequence of CDRL2 of 411D07 using Kabat 107 411D07 Amino QQYYSNRS CDRL3 acid (Kabat) sequence of CDRL3 of 411D07 using Kabat 108 411D07- Amino DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKS Light acid GQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQTEDVAVY chain sequence YCQQYYSNRSFGQGTKLEIK variable of region V_(L) of 411D07 109 411D07- Nucleic GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG Light acid GCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATA chain sequence CAGCTCCAACAATAAGAATTACTTAGCTTGGTACCAGCAGAAATCA variable of GGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAAT region V_(L) CCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTT of CACTCTCACCATCAGCAGCCTGCAGACTGAAGATGTGGCAGTTTAT 411D07 TACTGTCAGCAATATTATAGTAATCGCAGTTTTGGCCAGGGGACCA AGCTGGAGATCAAAC 110 411D07- Amino DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKS full acid GQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQTEDVAVY light sequence YCQQYYSNRSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV chain of CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL sequence 411D07 TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 111 411D07- Nucleic GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG full acid GCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATA light sequence CAGCTCCAACAATAAGAATTACTTAGCTTGGTACCAGCAGAAATCA chain of GGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAAT sequence 411D07 CCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTT light CACTCTCACCATCAGCAGCCTGCAGACTGAAGATGTGGCAGTTTAT chain TACTGTCAGCAATATTATAGTAATCGCAGTTTTGGCCAGGGGACCA AGCTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTT CCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTG TGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGA AGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGAC CGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTG ACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCG AAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAA CCGGGGCGAGTGT 112 385F01- Amino GFTFSSYW CDRH1 acid (IMGT) sequence of CDRH1 of 385F01 using IMGT 113 385F01- Amino IKEDGSEK CDRH2 acid (IMGT) sequence of CDRH2 of 385F01 using IMGT 114 385F01- Amino ARNRLYSDFLDN CDRH3 acid (IMGT) sequence of CDRH3 of 385F01 using IMGT 115 385F01- Amino SYWMS CDRH1 acid (Kabat) sequence of CDRH1 of 385F01 using Kabat 116 385F01 Amino NIKEDGSEKYYVDSVKG CDRH2 acid (Kabat) sequence of CDRH2 of 385F01 using Kabat 117 385F01- Amino NRLYSDFLDN CDRH3 acid (Kabat) sequence of CDRH3 of 385F01 using Kabat 118 385F01- Amino EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE Heavy acid WVANIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTS chain sequence VYYCARNRLYSDFLDNWGQGTLVTVSS variable of region V_(H) of 385F01 119 385F01- Nucleic GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG Heavy acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAG chain sequence CTATTGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG variable of TGGGTGGCCAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCG region V_(H) ACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA of CTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCT 385F01 GTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACA ACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG 120 385F01- Amino EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLE full acid WVANIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTS heavy sequence VYYCARNRLYSDFLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTS chain of GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL sequence 385F01 SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP heavy CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK chain FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 121 385F01- Nucleic GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG full acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAG heavy sequence CTATTGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG chain of TGGGTGGCCAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCG sequence 385F01 ACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAA heavy CTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCT chain GTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACA ACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAA GGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCT GGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCG AGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGT GCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTG TCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCT ACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA GAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCT TGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCC CCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGT GACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCA AGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGT GCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAG TGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCA TCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACT GCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACC TGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGG AGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTG GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGA TGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCT GAGCCCCGGCAAG 122 385F01 Amino QGVSSW CDRL1 acid (IMGT) sequence of CDRL1 of 385F01 using IMGT 123 385F01- Amino GAS CDRL2 acid (IMGT) sequence of CDRL2 of 385F01 using IMGT 124 385F01- Amino QQANSIPFT CDRL3 acid (IMGT) sequence of CDRL3 of 385F01 using IMGT 125 385F01- Amino RASQGVSSWLA CDRL1 acid (Kabat) sequence of CDRL1 of 385F01 using Kabat 126 385F01 Amino GASSLQS CDRL2 acid (Kabat) sequence of CDRL2 of 385F01 using Kabat 127 385F01- Amino QQANSIPFT CDRL3 acid (Kabat) sequence of CDRL3 of 385F01 using Kabat 128 385F01- Amino DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKL Light acid LIYGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQAN chain sequence SIPFTFGPGTKVDIK variable of region V_(L) of 385F01 129 385F01- Nucleic GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCG Light acid GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAG chain sequence CTGGTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTC variable of CTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGAT region V_(L) TCAGCGGCAGTGGATCTGGGACAGAGTTCATTCTCACCATCAGCAG of CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAAC 385F01 AGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC 130 385F01- Amino DIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKL full acid LIYGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQAN light sequence SIPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain of FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence 385F01 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 131 385F01- Nucleic GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCG full acid GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAG light sequence CTGGTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTC chain of CTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGAT sequence 385F01 TCAGCGGCAGTGGATCTGGGACAGAGTTCATTCTCACCATCAGCAG light CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAAC chain AGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 132 413D08 Amino GFTFRIYG CDRH1 acid (IMGT) sequence of CDRH1 of 413D08 using IMGT 133 413D08- Amino IWYDGSNK CDRH2 acid (IMGT) sequence of CDRH2 of 413D08 using IMGT 134 413D08- Amino ARDMDYFGMDV CDRH3 acid (IMGT) sequence of CDRH3 of 413D08 using IMGT 135 413D08- Amino IYGMH CDRH1 acid (Kabat) sequence of CDRH1 of 413D08 using Kabat 136 413D08 Amino VIWYDGSNKYYADSVKG CDRH2 acid (Kabat) sequence of CDRH2 of 413D08 using Kabat 137 413D08- Amino DMDYFGMDV CDRH3 acid (Kabat) sequence of CDRH3 of 413D08 using Kabat 138 413D08- Amino QVQLVESGGGVVQPGRSLRLSCAASGFTFRIYGMHWVRQAPGKGLE Heavy acid WVAVIWYDGSNKYYADSVKGRFTISRDNSDNTLYLQMNSLRAEDTA chain sequence VYYCARDMDYFGMDVWGQGTTVTVSS variable of region V_(H) of 413D08 139 413D08- Nucleic CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA Heavy acid GGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCCGTAT chain sequence TTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG variable of TGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCTG region V_(H) ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCGACAA of CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCT 413D08 GTGTATTACTGTGCGAGAGATATGGACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTCTCCTCAG 140 413D08- Amino QVQLVESGGGVVQPGRSLRLSCAASGFTFRIYGMHWVRQAPGKGLE full acid WVAVIWYDGSNKYYADSVKGRFTISRDNSDNTLYLQMNSLRAEDTA heavy sequence VYYCARDMDYFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSG chain of GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS sequence 413D08 SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC heavy PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF chain NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 141 413D08- Nucleic CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGA full acid GGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCCGTAT heavy sequence TTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAG chain of TGGGTGGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCTG sequence 413D08 ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCGACAA heavy CACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCT chain GTGTATTACTGTGCGAGAGATATGGACTACTTCGGTATGGACGTCT GGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGG CCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGC GGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGC CTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCA CACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCC TCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACA TCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAA GGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGT CCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCC CAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGAC CTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTC AATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGC CTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCT GACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGC AAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCT CCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCC CCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGT CTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGT CCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCT GGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGAC AAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGC ACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAG CCCCGGCAAG 142 413D08- Amino QGIRND CDRL1 acid (IMGT) sequence of CDRL1 of 413D08 using IMGT 143 413D08- Amino AAS CDRL2 acid (IMGT) sequence of CDRL2 of 413D08 using IMGT 144 413D08 Amino LQHNSYPRT CDRL3 acid (IMGT) sequence of CDRL3 of 413D08 using IMGT 145 413D08- Amino RASQGIRNDLG CDRL1 acid (Kabat) sequence of CDRL1 of 413D08 using Kabat 146 413D08- Amino AASSLQS CDRL2 acid (Kabat) sequence of CDRL2 of 413D08 using Kabat 147 413D08 Amino LQHNSYPRT CDRL3 acid (Kabat) sequence of CDRL3 of 413D08 using Kabat 148 413D08- Amino DLQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKR Light acid LIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHN chain sequence SYPRTFGQGTKVEIK variable of region V_(L) of 413D08 149 413D08- Nucleic GACCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG Light acid GAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAA chain sequence TGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGC variable of CTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGT region V_(L) TCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAG of CCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAAT 413D08 AGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC 150 413D08- Amino DLQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKR full acid LIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHN light sequence SYPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain of FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence 413D08 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 151 413D08- Nucleic GACCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG full acid GAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAA light sequence TGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGC chain of CTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGT sequence 413D08 TCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAG light CCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAAT chain AGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 152 386H03- Amino GGSISSSDW CDRH1 acid (IMGT) sequence of CDRH1 of 386H03 using IMGT 153 386H03- Amino IFHSGNT CDRH2 acid (IMGT) sequence of CDRH2 of 386H03 using IMGT 154 386H03- Amino VRDGSGSY CDRH3 acid (IMGT) sequence of CDRH3 of 386H03 using IMGT 155 386H03- Amino SSDWWS CDRH1 acid (Kabat) sequence of CDRH1 of 386H03 using Kabat 156 386H03 Amino EIFHSGNTNYNPSLKS CDRH2 acid (Kabat) sequence of CDRH2 of 386H03 using Kabat 157 386H03- Amino DGSGSY CDRH3 acid (Kabat) sequence of CDRH3 of 386H03 using Kabat 158 386H03- Amino QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGL Heavy acid EWIGEIFHSGNTNYNPSLKSRVTISVDKSKNQISLRLNSVTAADTA chain sequence VYYCVRDGSGSYWGQGTLVTVSS variable of region V_(H) of 386H03 159 386H03- Nucleic CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG Heavy acid GGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAG chain sequence TAGTGACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTG variable of GAGTGGATTGGGGAAATCTTTCATAGTGGGAACACCAACTACAACC region V_(H) CGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAA of CCAGATCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCC 386H03 GTGTATTACTGTGTGAGAGATGGTTCGGGGAGTTACTGGGGCCAGG GAACCCTGGTCACCGTCTCCTCAG 160 386H03- Amino QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGL full acid EWIGEIFHSGNTNYNPSLKSRVTISVDKSKNQISLRLNSVTAADTA heavy sequence VYYCVRDGSGSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA chain of ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV sequence 386H03 TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP heavy ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY chain VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 161 386H03- Nucleic CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGG full acid GGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAG heavy sequence TAGTGACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTG chain of GAGTGGATTGGGGAAATCTTTCATAGTGGGAACACCAACTACAACC sequence 386H03 CGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAA heavy CCAGATCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCC chain GTGTATTACTGTGTGAGAGATGGTTCGGGGAGTTACTGGGGCCAGG GAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGT GTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCC GCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCG TGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCC TGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTG ACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACG TGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACC CAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCT GAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCA AGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGT GGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGG AACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCT GCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCC AACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCA AGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAG GGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAA GGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCC AGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGA CGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGG TGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCC TGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAA G 162 386H03- Amino QSVLYSSNNKNY CDRL1 acid (IMGT) sequence of CDRL1 of 386H03 using IMGT 163 386H03 Amino WAS CDRL2 acid (IMGT) sequence of CDRL2 of 386H03 using IMGT 164 386H03- Amino QQYYSTRS CDRL3 acid (IMGT) sequence of CDRL3 of 386H03 using IMGT 165 386H03- Amino KSSQSVLYSSNNKNYLA CDRL1 acid (Kabat) sequence of CDRL1 of 386H03 using Kabat 166 386H03- Amino WASTRES CDRL2 acid (Kabat) sequence of CDRL2 of 386H03 using Kabat 167 386H03- Amino QQYYSTRS CDRL3 acid (Kabat) sequence of CDRL3 of 386H03 using Kabat 168 386H03- Amino DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKP Light acid GQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY chain sequence YCQQYYSTRSFGQGTKLEIK variable of region V_(L) of 386H03 169 386H03- Nucleic GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG Light acid GCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATA chain sequence CAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCA variable of GGACAGCCTCCTAAACTGCTCATTTACTGGGCATCTACCCGGGAAT region V_(L) CCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTT of CACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTAT 386H03 TACTGTCAGCAATATTATAGTACTCGCAGTTTTGGCCAGGGGACCA AGCTGGAGATCAAAC 170 386H03- Amino DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKP full acid GQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY light sequence YCQQYYSTRSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV chain of CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL sequence 386H03 TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 171 386H03- Nucleic GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG full acid GCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATA light sequence CAGCTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCA chain of GGACAGCCTCCTAAACTGCTCATTTACTGGGCATCTACCCGGGAAT sequence 386H03 CCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTT light CACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTAT chain TACTGTCAGCAATATTATAGTACTCGCAGTTTTGGCCAGGGGACCA AGCTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTT CCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTG TGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGA AGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGAC CGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTG ACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCG AAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAA CCGGGGCGAGTGT 172 389A03- Amino GGSISSSSYY CDRH1 acid (IMGT) sequence of CDRH1 of 389A03 using IMGT 173 389A03- Amino IYSTGYT CDRH2 acid (IMGT) sequence of CDRH2 of 389A03 using IMGT 174 389A03- Amino AISTAAGPEYFHR CDRH3 acid (IMGT) sequence of CDRH3 of 389A03 using IMGT 175 389A03 Amino SSSYYCG CDRH1 acid (Kabat) sequence of CDRH1 of 389A03 using Kabat 176 389A03 Amino SIYSTGYTYYNPSLKS CDRH2 acid (Kabat) sequence of CDRH2 of 389A03 using Kabat 177 389A03- Amino STAAGPEYFHR CDRH3 acid (Kabat) sequence of CDRH3 of 389A03 using Kabat 178 389A03 Amino QLQESGPGLVKPSETLSLTCTVSGGSISSSSYYCGWIRQPPGKGLD Heavy acid WIGSIYSTGYTYYNPSLKSRVTISIDTSKNQFSCLILTSVTAADTA chain sequence VYYCAISTAAGPEYFHRWGQGTLVTVSS variable of region V_(H) of 389A03 179 389A03- Nucleic CAGCTGCAGGAGTCGGGCCCAGGCCTGGTGAAGCCTTCGGAGACCC Heavy acid TGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAG chain sequence TTATTACTGCGGCTGGATCCGCCAGCCCCCTGGGAAGGGGCTGGAC variable of TGGATTGGGAGTATCTATTCTACTGGGTACACCTACTACAACCCGT region V_(H) CCCTCAAGAGTCGAGTCACCATTTCCATAGACACGTCCAAGAACCA of GTTCTCATGCCTGATACTGACCTCTGTGACCGCCGCAGACACGGCT 389A03 GTGTATTACTGTGCGATAAGTACAGCAGCTGGCCCTGAATACTTCC ATCGCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG 180 389A03- Amino QLQESGPGLVKPSETLSLTCTVSGGSISSSSYYCGWIRQPPGKGLD full acid WIGSIYSTGYTYYNPSLKSRVTISIDTSKNQFSCLILTSVTAADTA heavy sequence VYYCAISTAAGPEYFHRWGQGTLVTVSSASTKGPSVFPLAPSSKST chain of SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS sequence 389A03 LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP heavy PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV chain KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 181 389A03 Nucleic CAGCTGCAGGAGTCGGGCCCAGGCCTGGTGAAGCCTTCGGAGACCC full acid TGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAG heavy sequence TTATTACTGCGGCTGGATCCGCCAGCCCCCTGGGAAGGGGCTGGAC chain of TGGATTGGGAGTATCTATTCTACTGGGTACACCTACTACAACCCGT sequence 389A03 CCCTCAAGAGTCGAGTCACCATTTCCATAGACACGTCCAAGAACCA heavy GTTCTCATGCCTGATACTGACCTCTGTGACCGCCGCAGACACGGCT chain GTGTATTACTGTGCGATAAGTACAGCAGCTGGCCCTGAATACTTCC ATCGCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCAGCAC CAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACC TCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCC CCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGG AGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCC CTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGA CCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGA CAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCC CCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGT TCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGA AGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTG AAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGA CCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTC CGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTAC AAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGA CCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACAC ACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTG ACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAAT GGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCC TGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACA GTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCG TGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTC CCTGAGCCCCGGCAAG 182 389A03- Amino QSVLYSSNSKNF CDRL1 acid (IMGT) sequence of CDRL1 of 389A03 using IMGT 183 389A03 Amino WAS CDRL2 acid (IMGT) sequence of CDRL2 of 389A03 using IMGT 184 389A03- Amino QQYYSTPRT CDRL3 acid (IMGT) sequence of CDRL3 of 389A03 using IMGT 185 389A03- Amino KSSQSVLYSSNSKNFLA CDRL1 acid (Kabat) sequence of CDRL1 of 389A03 using Kabat 186 389A03- Amino WASTRGS CDRL2 acid (Kabat) sequence of CDRL2 of 389A03 using Kabat 187 389A03- Amino QQYYSTPRT CDRL3 acid (Kabat) sequence of CDRL3 of 389A03 using Kabat 188 389A03 Amino DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNFLAWYQQKP Light acid GQPPKLFIYWASTRGSGVPDRISGSGSGTDFNLTISSLQAEDVAVY chain sequence YCQQYYSTPRTFGQGTKVEIK variable of region V_(L) of 389A03 189 389A03- Nucleic GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG Light acid GCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATA chain sequence CAGCTCCAACAGTAAGAACTTCTTAGCTTGGTACCAGCAGAAACCG variable of GGACAGCCTCCTAAGCTGTTCATTTACTGGGCATCTACCCGGGGAT region V_(L) CCGGGGTCCCTGACCGAATCAGTGGCAGCGGGTCTGGGACAGATTT of CAATCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTAT 389A03 TACTGTCAACAATATTATAGTACTCCTCGGACGTTCGGCCAAGGGA CCAAGGTGGAGATCAAAC 190 389A03- Amino DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNFLAWYQQKP full acid GQPPKLFIYWASTRGSGVPDRISGSGSGTDFNLTISSLQAEDVAVY light sequence YCQQYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV chain of VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST sequence 389A03 LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 191 389A03- Nucleic acid GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGG full Sequence of GCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATA light 389A03 CAGCTCCAACAGTAAGAACTTCTTAGCTTGGTACCAGCAGAAACCG chain light chain GGACAGCCTCCTAAGCTGTTCATTTACTGGGCATCTACCCGGGGAT sequence CCGGGGTCCCTGACCGAATCAGTGGCAGCGGGTCTGGGACAGATTT CAATCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTAT TACTGTCAACAATATTATAGTACTCCTCGGACGTTCGGCCAAGGGA CCAAGGTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCAT CTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTC GTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGT GGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGT GACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACC CTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCT GCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTT CAACCGGGGCGAGTGT 192 Human IGHG* Heavy gcttccaccaagggcccatccgtcttccccctggcgccctgctcca IgG4 01 Chain ggagcacctccgagagcacagccgccctgggctgcctggtcaagga heavy & Constant ctacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg chain IGHG4 Region accagcggcgtgcacaccttcccggctgtcctacagtcctcaggac constant *04 Nucleotide tctactccctcagcagcgtggtgaccgtgccctccagcagcttggg region Sequence cacgaagacctacacctgcaacgtagatcacaagcccagcaacacc #1 aaggtggacaagagagttgagtccaaatatggtcccccatgcccat catgcccagcacctgagttcctggggggaccatcagtcttcctgtt ccccccaaaacccaaggacactctcatgatctcccggacccctgag gtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtcc agttcaactggtacgtggatggcgtggaggtgcataatgccaagac aaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagc gtcctcaccgtcctgcaccaggactggctgaacggcaaggagtaca agtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaac catctccaaagccaaagggcagccccgagagccacaggtgtacacc ctgcccccatcccaggaggagatgaccaagaaccaggtcagcctga cctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtg ggagagcaatgggcagccggagaacaactacaagaccacgcctccc gtgctggactccgacggctccttcttcctctacagcaggctaaccg tggacaagagcaggtggcaggaggggaatgtcttctcatgctccgt gatgcatgaggctctgcacaaccactacacacagaagagcctctcc ctgtctctgggtaaa 193 Heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL Chain TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT Constant KVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPE Region VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS Amino VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT Acid LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP Sequence VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 194 Human IGHG* Heavy gcttccaccaagggcccatccgtcttccccctggcgccctgctcca IgG4 02 Chain ggagcacctccgagagcacagccgccctgggctgcctggtcaagga heavy Constant ctacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg chain Region accagcggcgtgcacaccttcccggctgtcctacagtcctcaggac constant Nucleotide tctactccctcagcagcgtggtgaccgtgccctccagcagcttggg region Sequence cacgaagacctacacctgcaacgtagatcacaagcccagcaacacc #2 aaggtggacaagagagttgagtccaaatatggtcccccgtgcccat catgcccagcacctgagttcctggggggaccatcagtcttcctgtt ccccccaaaacccaaggacactctcatgatctcccggacccctgag gtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtcc agttcaactggtacgtggatggcgtggaggtgcataatgccaagac aaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagc gtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtaca agtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaac catctccaaagccaaagggcagccccgagagccacaggtgtacacc ctgcccccatcccaggaggagatgaccaagaaccaggtcagcctga cctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtg ggagagcaatgggcagccggagaacaactacaagaccacgcctccc gtgctggactccgacggctccttcttcctctacagcaggctaaccg tggacaagagcaggtggcaggaggggaatgtcttctcatgctccgt gatgcatgaggctctgcacaaccactacacgcagaagagcctctcc ctgtctctgggtaaa 195 Heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL Chain TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT Constant KVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPE Region VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS Amino VLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT Acid LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP Sequence VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 196 Human IGHG* Heavy gcttccaccaagggcccatccgtcttccccctggcgccctgctcca IgG4 03 Chain ggagcacctccgagagcacagccgccctgggctgcctggtcaagga heavy Constant ctacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg chain Region accagcggcgtgcacaccttcccggctgtcctacagtcctcaggac constant Nucleotide tctactccctcagcagcgtggtgaccgtgccctccagcagcttggg region Sequence cacgaagacctacacctgcaacgtagatcacaagcccagcaacacc #3 aaggtggacaagagagttgagtccaaatatggtcccccatgcccat catgcccagcacctgagttcctggggggaccatcagtcttcctgtt ccccccaaaacccaaggacactctcatgatctcccggacccctgag gtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtcc agttcaactggtacgtggatggcgtggaggtgcataatgccaagac aaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagc gtcctcaccgtcctgcaccaggactggctgaacggcaaggagtaca agtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaac catctccaaagccaaagggcagccccgagagccacaggtgtacacc ctgcccccatcccaggaggagatgaccaagaaccaggtcagcctga cctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtg ggagagcaatgggcagccggagaacaactacaagaccacgcctccc gtgctggactccgacggctccttcttcctctacagcaagctcaccg tggacaagagcaggtggcaggaggggaacgtcttctcatgctccgt gatgcatgaggctctgcacaaccactacacgcagaagagcctctcc ctgtctctgggtaaa 197 Heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL Chain TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT Constant KVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPE Region VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS Amino VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT Acid LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP Sequence VLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 198 IgG4 - Heavy gcctccaccaagggcccatccgtcttccccctggcgccctgctcca heavy IgG4- Chain ggagcacctccgagagcacggccgccctgggctgcctggtcaagga chain PE Constant ctacttccccgaaccagtgacggtgtcgtggaactcaggcgccctg constant Region accagcggcgtgcacaccttcccggctgtcctacagtcctcaggac region- Nucleotide tctactccctcagcagcgtggtgaccgtgccctccagcagcttggg IgG4-PE Sequence- cacgaagacctacacctgcaacgtagatcacaagcccagcaacacc Synthetic aaggtggacaagagagttgagtccaaatatggtcccccatgcccac Version catgcccagcgcctgaatttgaggggggaccatcagtcttcctgtt A ccccccaaaacccaaggacactctcatgatctcccggacccctgag gtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtcc agttcaactggtacgtggatggcgtggaggtgcataatgccaagac aaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagc gtcctcaccgtcctgcaccaggactggctgaacggcaaggagtaca agtgcaaggtctccaacaaaggcctcccgtcatcgatcgagaaaac catctccaaagccaaagggcagccccgagagccacaggtgtacacc ctgcccccatcccaggaggagatgaccaagaaccaggtcagcctga cctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtg ggagagcaatgggcagccggagaacaactacaagaccacgcctccc gtgctggactccgacggatccttcttcctctacagcaggctaaccg tggacaagagcaggtggcaggaggggaatgtcttctcatgctccgt gatgcatgaggctctgcacaaccactacacacagaagagcctctcc ctgtctctgggtaaa 199 IgG4 Heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL heavy Chain TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT chain Constant KVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPE constant Region VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS region- Amino VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT IgG4-PE Acid LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP Sequence- VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS Encoded LSLGK by Synthetic Version A, B & C (Two residues that differ from the wild-type sequence are identified in bold) 200 IgG4 Heavy Gcctccaccaagggacctagcgtgttccctctcgccccctgttcca heavy Chain ggtccacaagcgagtccaccgctgccctcggctgtctggtgaaaga chain Constant ctactttcccgagcccgtgaccgtctcctggaatagcggagccctg constant Region acctccggegtgcacacatttcccgccgtgctgcagagcagcggac region- Nucleotide tgtatagcctgagcagcgtggtgaccgtgcccagctccagcctcgg IgG4-PE Sequence- caccaaaacctacacctgcaacgtggaccacaagccctccaacacc Synthetic aaggtggacaagcgggtggagagcaagtacggccccccttgccctc Version cttgtcctgcccctgagttcgagggaggaccctccgtgttcctgtt B tccccccaaacccaaggacaccctgatgatctcccggacacccgag gtgacctgtgtggtcgtggacgtcagccaggaggaccccgaggtgc agttcaactggtatgtggacggcgtggaggtgcacaatgccaaaac caagcccagggaggagcagttcaattccacctacagggtggtgagc gtgctgaccgtcctgcatcaggattggctgaacggcaaggagtaca agtgcaaggtgtccaacaagggactgcccagctccatcgagaagac catcagcaaggctaagggccagccgagggagccccaggtgtatacc ctgcctcctagccaggaagagatgaccaagaaccaagtgtccctga cctgcctggtgaagggattctacccctccgacatcgccgtggagtg ggagagcaatggccagcccgagaacaactacaaaacaacccctccc gtgctcgatagcgacggcagcttctttctctacagccggctgacag tggacaagagcaggtggcaggagggcaacgtgttctcctgttccgt gatgcacgaggccctgcacaatcactacacccagaagagcctctcc ctgtccctgggcaag 201 IgG4 Heavy gccagcaccaagggcccttccgtgttccccctggccccttgcagca heavy Chain ggagcacctccgaatccacagctgccctgggctgtctggtgaagga chain Constant ctactttcccgagcccgtgaccgtgagctggaacagcggcgctctg constant Region acatccggcgtccacacctttcctgccgtcctgcagtcctccggcc region- Nucleotide tctactccctgtcctccgtggtgaccgtgcctagctcctccctcgg IgG4-PE Sequence- caccaagacctacacctgtaacgtggaccacaaaccctccaacacc Synthetic aaggtggacaaacgggtcgagagcaagtacggccctccctgccctc Version cttgtcctgcccccgagttcgaaggcggacccagcgtgttcctgtt C ccctcctaagcccaaggacaccctcatgatcagccggacacccgag gtgacctgcgtggtggtggatgtgagccaggaggaccctgaggtcc agttcaactggtatgtggatggcgtggaggtgcacaacgccaagac aaagccccgggaagagcagttcaactccacctacagggtggtcagc gtgctgaccgtgctgcatcaggactggctgaacggcaaggagtaca agtgcaaggtcagcaataagggactgcccagcagcatcgagaagac catctccaaggctaaaggccagccccgggaacctcaggtgtacacc ctgcctcccagccaggaggagatgaccaagaaccaggtgagcctga cctgcctggtgaagggattctacccttccgacatcgccgtggagtg ggagtccaacggccagcccgagaacaattataagaccacccctccc gtcctcgacagcgacggatccttctttctgtactccaggctgaccg tggataagtccaggtggcaggaaggcaacgtgttcagctgctccgt gatgcacgaggccctgcacaatcactacacccagaagtccctgagc ctgtccctgggaaag 202 IgG4 Heavy gcctccaccaagggcccatccgtcttccccctggcgccctgctcca heavy Chain ggagcacctccgagagcacggccgccctgggctgcctggtcaagga chain Constant ctacttccccgaaccagtgacggtgtcgtggaactcaggcgccctg constant Region accagcggcgtgcacaccttcccggctgtcctacagtcctcaggac region Nucleotide tctactccctcagcagcgtggtgaccgtgccctccagcagcttggg Sequence- cacgaagacctacacctgcaacgtagatcacaagcccagcaacacc Synthetic aaggtggacaagagagttgagtccaaatatggtcccccatgcccac Version catgcccagcgcctccagttgcggggggaccatcagtcttcctgtt D ccccccaaaacccaaggacactctcatgatctcccggacccctgag gtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtcc agttcaactggtacgtggatggcgtggaggtgcataatgccaagac aaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagc gtcctcaccgtcctgcaccaggactggctgaacggcaaggagtaca agtgcaaggtctccaacaaaggcctcccgtcatcgatcgagaaaac catctccaaagccaaagggcagccccgagagccacaggtgtacacc ctgcccccatcccaggaggagatgaccaagaaccaggtcagcctga cctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtg ggagagcaatgggcagccggagaacaactacaagaccacgcctccc gtgctggactccgacggatccttcttcctctacagcaggctaaccg tggacaagagcaggtggcaggaggggaatgtcttctcatgctccgt gatgcatgaggctctgcacaaccactacacacagaagagcctctcc ctgtctctgggtaaa 203 Heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL Chain TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT Constant KVDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPE Region VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS Amino VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT Acid LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP Sequence- VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS encoded LSLGK by Synthetic Version D 204 Disabled Disabled Heavy gcctccaccaagggcccatcggtcttccccctggcaccctcctcca Human IGHG1 Chain agagcacctctgggggcacagcggccctgggctgcctggtcaagga IgG1 Constant ctacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg heavy Region accagcggcgtgcacaccttcccggctgtcctacagtcctcaggac chain Nucleotide tctactccctcagcagcgtggtgaccgtgccctccagcagcttggg constant Sequence cacccagacctacatctgcaacgtgaatcacaagcccagcaacacc region aaggtggacaagaaagtggagcccaaatcttgtgacaaaactcaca catgcccaccgtgcccagcacctgaactcgcgggggcaccgtcagt cttcctcttccccccaaaacccaaggacaccctcatgatctcccgg acccctgaggtcacatgcgtggtggtggacgtgagccacgaagacc ctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataa tgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggca aggagtacaagtgcaaggtctccaacaaagccctcccagcccccat cgagaaaaccatctccaaagccaaagggcagccccgagaaccacag gtgtacaccctgcccccatcccgggatgagctgaccaagaaccagg tcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc cgtggagtgggagagcaatgggcagccggagaacaactacaagacc acgcctcccgtgctggactccgacggctccttcttcctctacagca agctcaccgtggacaagagcaggtggcagcaggggaacgtcttctc atgctccgtgatgcatgaggctctgcacaaccactacacgcagaag agcctctccctgtctccgggtaaa 205 Heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL Chain TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT Constant KVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISR Region TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR Amino VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ Acid VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT Sequence TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK (Two SLSLSPGK residues that differ from the wild-type sequence are identified in bold) 206 Human IGKC* Cκ cgtacggtggccgctccctccgtgttcatcttcccaccttccgacg Cκ 01 Light agcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaa constant Chain cttctacccccgcgaggccaaggtgcagtggaaggtggacaacgcc region Constant ctgcagtccggcaactcccaggaatccgtgaccgagcaggactcca Region aggacagcacctactccctgtcctccaccctgaccctgtccaaggc Nucleotide cgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccag Sequence ggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt 207 Cκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA Light LOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ Chain GLSSPVTKSFNRGEC Constant Region Amino Acid Sequence 208 Human IGKC* Cκ cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg Cκ 02 Light agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataa constant Chain cttctatcccagagaggccaaagtacagtggaaggtggataacgcc region Constant ctccaatcgggtaactcccaggagagtgtcacagagcaggagagca Region aggacagcacctacagcctcagcagcaccctgacgctgagcaaagc Nucleotide agactacgagaaacacaaagtctacgccggcgaagtcacccatcag Sequence ggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 209 Cκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA Light LQSGNSQESVTEQESKDSTYSLSSTLTLSKADYEKHKVYAGEVTHQ Chain GLSSPVTKSFNRGEC Constant Region Amino Acid Sequence 210 Human IGKC* Cκ cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg Cκ 03 Light agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataa constant Chain cttctatcccagagaggccaaagtacagcggaaggtggataacgcc region Constant ctccaatcgggtaactcccaggagagtgtcacagagcaggagagca Region aggacagcacctacagcctcagcagcaccctgacgctgagcaaagc Nucleotide agactacgagaaacacaaagtctacgcctgcgaagtcacccatcag Sequence ggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 211 Cκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQRKVDNA Light LQSGNSQESVTEQESKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ Chain GLSSPVTKSFNRGEC Constant Region Amino Acid Sequence 212 Human IGKC* Cκ cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg Cκ 04 Light agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataa constant Chain cttctatoccagagaggccaaagtacagtggaaggtggataacgcc region Constant 01 Region ctccaatcgggtaactcccaggagagtgtcacagagcaggacagca Nucleotide aggacagcacctacagcctcagcagcaccctgacgctgagcaaagc Sequence agactacgagaaacacaaactctacgcctgcgaagtcacccatcag ggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 213 Cκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA Light LOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQ Chain GLSSPVTKSFNRGEC Constant Region Amino Acid Sequence 214 Human IGKC* Cκ cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatg Cκ 05 Light agcagttgaaatctggaactgcctctgttgtgtgcctgctgaataa constant Chain cttctatcccagagaggccaaagtacagtggaaggtggataacgcc region Constant ctccaatcgggtaactcccaggagagtgtcacagagcaggacagca Region aggacagcacctacagcctcagcaacaccctgacgctgagcaaagc Nucleotide agactacgagaaacacaaagtctacgcctgcgaagtcacccatcag Sequence ggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc 215 Cκ RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA Light LQSGNSQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQ Chain GLSSPVTKSFNRGEC Constant Region Amino Acid Sequence 216 Human IGCλ1 Cλ cccaaggccaaccccacggtcactctgttcccgccctcctctgagg Cλ *01 Light agctccaagccaacaaggccacactagtgtgtctgatcagtgactt constant Chain ctacccgggagctgtgacagtggcttggaaggcagatggcagcccc region Constant gtcaaggcgggagtggagacgaccaaaccctccaaacagagcaaca Region acaagtacgcggccagcagctacctgagcctgacgcccgagcagtg Nucleotide gaagtcccacagaagctacagctgccaggtcacgcatgaagggagc Sequence accgtggagaagacagtggcccctacagaatgttca 217 Cλ PKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSP Light VKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGS Chain TVEKTVAPTECS Constant Region Amino Acid Sequence 218 Human IGCλ1 Cλ ggtcagcccaaggccaaccccactgtcactctgttcccgccctcct Cλ *02 Light ctgaggagctccaagccaacaaggccacactagtgtgtctgatcag constant Chain tgacttctacccgggagctgtgacagtggcctggaaggcagatggc region Constant agccccgtcaaggcgggagtggagaccaccaaaccctccaaacaga Region gcaacaacaagtacgcggccagcagctacctgagcctgacgcccga Nucleotide gcagtggaagtcccacagaagctacagctgccaggtcacgcatgaa Sequence gggagcaccgtggagaagacagtggcccctacagaatgttca 219 Cλ GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADG Light SPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE Chain GSTVEKTVAPTECS Constant Region Amino Acid Sequence 220 Human IGCλ2 Cλ ggtcagcccaaggccaaccccactgtcactctgttcccgccctcct Cλ *01 Light ctgaggagctccaagccaacaaggccacactagtgtgtctgatcag constant Chain tgacttctacccgggagctgtgacagtggcctggaaggcagatggc region Constant agccccgtcaaggcgggagtggagaccaccaaaccctccaaacaga Region gcaacaacaagtacgcggccagcagctacctgagcctgacgcccga Nucleotide gcagtggaagtcccacagaagctacagctgccaggtcacgcatgaa Sequence- gggagcaccgtggagaagacagtggcccctacagaatgttca Version A 221 Cλ ggccagcctaaggccgctccttctgtgaccctgttccccccatcct Light ccgaggaactgcaggctaacaaggccaccctcgtgtgcctgatcag Chain cgacttctaccctggcgccgtgaccgtggcctggaaggctgatagc Constant tctcctgtgaaggccggcgtggaaaccaccaccccttccaagcagt Region ccaacaacaaatacgccgcctcctcctacctgtccctgacccctga Nucleotide gcagtggaagtcccaccggtcctacagctgccaagtgacccacgag Sequence- ggctccaccgtggaaaagaccgtggctcctaccgagtgctcc Version B 222 Cλ ggccagcctaaagctgcccccagcgtcaccctgtttcctccctcca Light gcgaggagctccaggccaacaaggccaccctcgtgtgcctgatctc Chain cgacttctatcccggcgctgtgaccgtggcttggaaagccgactcc Constant agccctgtcaaagccggcgtggagaccaccacaccctccaagcagt Region ccaacaacaagtacgccgcctccagctatctctccctgacccctga Nucleotide gcagtggaagtcccaccggtcctactcctgtcaggtgacccacgag Sequence- ggctccaccgtggaaaagaccgtcgcccccaccgagtgctcc Version C 223 Cλ GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADG Light SPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE Chain GSTVEKTVAPTECS Constant Region Amino Acid Sequence- Encoded by Version A, B & C 224 Human IGCλ2 Cλ ggtcagcccaaggctgccccctcggtcactctgttcccgccctcct Cλ *02 Light ctgaggagcttcaagccaacaaggccacactggtgtgtctcataag constant & Chain tgacttctacccgggagccgtgacagtggcctggaaggcagatagc region IGLC2 Constant agccccgtcaaggcgggagtggagaccaccacaccctccaaacaaa *03 Region gcaacaacaagtacgcggccagcagctatctgagcctgacgcctga Nucleotide gcagtggaagtcccacagaagctacagctgccaggtcacgcatgaa Sequence gggagcaccgtggagaagacagtggcccctacagaatgttca 225 Cλ GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS Light SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE Chain GSTVEKTVAPTECS Constant Region Amino Acid Sequence 226 Human IGCλ3 Cλ cccaaggctgccccctcggtcactctgttcccaccctcctctgagg Cλ *01 Light agcttcaagccaacaaggccacactggtgtgtctcataagtgactt constant Chain ctacccgggagccgtgacagttgcctggaaggcagatagcagcccc region Constant gtcaaggcgggggtggagaccaccacaccctccaaacaaagcaaca Region acaagtacgcggccagcagctacctgagcctgacgcctgagcagtg Nucleotide gaagtcccacaaaagctacagctgccaggtcacgcatgaagggagc Sequence accgtggagaagacagttgcccctacggaatgttca 227 Cλ PKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSP Light VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGS Chain TVEKTVAPTECS Constant Region Amino Acid Sequence 228 Human IGCλ3 Cλ ggtcagcccaaggctgccccctcggtcactctgttcccaccctcct Cλ *02 Light ctgaggagcttcaagccaacaaggccacactggtgtgtctcataag constant Chain tgacttctacccggggccagtgacagttgcctggaaggcagatagc region Constant agccccgtcaaggcgggggtggagaccaccacaccctccaaacaaa Region gcaacaacaagtacgcggccagcagctacctgagcctgacgcctga Nucleotide gcagtggaagtcccacaaaagctacagctgccaggtcacgcatgaa Sequence gggagcaccgtggagaagacagtggcccctacggaatgttca 229 Cλ GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGPVTVAWKADS Light SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHE Chain GSTVEKTVAPTECS Constant Region Amino Acid Sequence 230 Human IGCλ3 Cλ ggtcagcccaaggctgccccctcggtcactctgttcccaccctcct Cλ *03 Light ctgaggagcttcaagccaacaaggccacactggtgtgtctcataag constant Chain tgacttctacccgggagccgtgacagtggcctggaaggcagatagc region Constant agccccgtcaaggcgggagtggagaccaccacaccctccaaacaaa Region gcaacaacaagtacgcggccagcagctacctgagcctgacgcctga Nucleotide gcagtggaagtcccacaaaagctacagctgccaggtcacgcatgaa Sequence gggagcaccgtggagaagacagtggcccctacagaatgttca 231 Cλ GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS Light SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHE Chain GSTVEKTVAPTECS Constant Region Amino Acid Sequence 232 Human IGCλ3 Cλ ggtcagcccaaggctgccccctcggtcactctgttcccgccctcct Cλ *04 Light ctgaggagcttcaagccaacaaggccacactggtgtgtctcataag constant Chain tgacttctacccgggagccgtgacagtggcctggaaggcagatagc region Constant agccccgtcaaggcgggagtggagaccaccacaccctccaaacaaa Region gcaacaacaagtacgcggccagcagctacctgagcctgacgcctga Nucleotide gcagtggaagtcccacagaagctacagctgccaggtcacgcatgaa Sequence gggagcaccgtggagaagacagtggcccctacagaatgttca 233 Cλ GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS Light SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE Chain GSTVEKTVAPTECS Constant Region Amino Acid Sequence 234 Human IGCλ6 Cλ ggtcagcccaaggctgccccatcggtcactctgttcccgccctcct Cλ *01 Light ctgaggagcttcaagccaacaaggccacactggtgtgcctgatcag constant Chain tgacttctacccgggagctgtgaaagtggcctggaaggcagatggc region Constant agccccgtcaacacgggagtggagaccaccacaccctccaaacaga Region gcaacaacaagtacgcggccagcagctacctgagcctgacgcctga Nucleotide gcagtggaagtcccacagaagctacagctgccaggtcacgcatgaa Sequence gggagcaccgtggagaagacagtggcccctgcagaatgttca 235 Cλ GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADG Light SPVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE Chain GSTVEKTVAPAECS Constant Region Amino Acid Sequence 236 Human IGLC7 Cλ ggtcagcccaaggctgccccatcggtcactctgttcccaccctcct Cλ *01 Light ctgaggagcttcaagccaacaaggccacactggtgtgtctcgtaag constant & Chain tgacttctacccgggagccgtgacagtggcctggaaggcagatggc region IGCλ7 Constant agccccgtcaaggtgggagtggagaccaccaaaccctccaaacaaa *02 Region gcaacaacaagtatgcggccagcagctacctgagcctgacgcccga Nucleotide gcagtggaagtcccacagaagctacagctgccgggtcacgcatgaa Sequence gggagcaccgtggagaagacagtggcccctgcagaatgctct 237 Cλ GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADG Light SPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHE Chain GSTVEKTVAPAECS Constant Region Amino Acid Sequence 238 413G05- Amino GFTFSDYY CDRH1 acid (IMGT) sequence of CDRH1 of 413G05 using IMGT 239 413G05 Amino ISTSGSTI CDRH2 acid (IMGT) sequence of CDRH2 of 413G05 using IMGT 240 413G05- Amino ARGITGTNFYHYGLGV CDRH3 acid (IMGT) sequence of CDRH3 of 413G05 using IMGT 241 413G05- Amino DYYMS CDRH1 acid (Kabat) sequence of CDRH1 of 413G05 using Kabat 242 413G05- Amino YISTSGSTIYYADSVKG CDRH2 acid (Kabat) sequence of CDRH2 of 413G05 using Kabat 243 413G05- Amino GITGTNFYHYGLGV CDRH3 acid (Kabat) sequence of CDRH3 of 413G05 using Kabat 244 413G05- Amino QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQVPGKGLE Heavy acid WVSYISTSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDAA chain sequence VYHCARGITGTNFYHYGLGVWGQGTTVTVSS variable of region V_(H) of 413G05 245 413G05- Nucleic CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAG Heavy acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGA chain sequence CTACTACATGAGCTGGATCCGCCAGGTTCCAGGGAAGGGGCTGGAG variable of TGGGTTTCATACATTAGTACTAGTGGTAGTACCATATACTACGCAG region V_(H) ACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAA of CTCACTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACGCGGCC 413G05 GTGTATCACTGTGCGAGAGGTATAACTGGAACTAACTTCTACCACT ACGGTTTGGGCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTC AG 246 413G05- Amino QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQVPGKGLE full acid WVSYISTSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDAA heavy sequence VYHCARGITGTNFYHYGLGVWGQGTTVTVSSASTKGPSVFPLAPSS chain of KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence 413G05 LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH heavy TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED chain PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 247 413G05- Nucleic CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAG full acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGA 11 sequence CTACTACATGAGCTGGATCCGCCAGGTTCCAGGGAAGGGGCTGGAG heavy of TGGGTTTCATACATTAGTACTAGTGGTAGTACCATATACTACGCAG chain 413G05 ACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAA sequence heavy CTCACTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACGCGGCC chain GTGTATCACTGTGCGAGAGGTATAACTGGAACTAACTTCTACCACT ACGGTTTGGGCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTC AGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGC AAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGG ACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCT GACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGC CTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGG GCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACAC CAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCAC ACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCG TGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCG GACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGAC CCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACA ACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCG GGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCA TCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCA GGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAG GTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCG CCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGAC CACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGC AAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCT CCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA GTCCCTGTCCCTGAGCCCCGGCAAG 248 413G05- Amino QGINSW CDRL1 acid (IMGT) sequence of CDRL1 of 413G05 using IMGT 249 413G05- Amino AAS CDRL2 acid (IMGT) sequence of CDRL2 of 413G05 using IMGT 250 413G05 Amino QQVNSFPLT CDRL3 acid (IMGT) sequence of CDRL3 of 413G05 using IMGT 251 413G05- Amino RASQGINSWLA CDRL1 acid (Kabat) sequence of CDRL1 of 413G05 using Kabat 252 413G05- Amino AASTLQS CDRL2 acid (Kabat) sequence of CDRL2 of 413G05 using Kabat 253 413G05- Amino QQVNSFPLT CDRL3 acid (Kabat) sequence of CDRL3 of 413G05 using Kabat 254 413G05- Amino DIQMTQSPSSVSASVGDRVTITCRASQGINSWLAWYQQKPGKAPKL Light acid LIYAASTLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQVN chain sequence SFPLTFGGGTKVEIK variable of region V_(L) of 413G05 255 413G05- Nucleic GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAG Light acid GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAACAG chain sequence CTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTC variable of CTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGT region V_(L) TCAGCGGCAGTGGGTCTGGGGCAGATTTCACTCTCACCATCAGCAG of CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGTTAAC 413G05 AGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC 256 413G05 Amino DIQMTQSPSSVSASVGDRVTITCRASQGINSWLAWYQQKPGKAPKL full acid LIYAASTLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQVN light sequence SFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain of FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence 413G05 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 257 413G05- Nucleic GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAG full acid GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAACAG light sequence CTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTC chain of CTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGT sequence 413G05 TCAGCGGCAGTGGGTCTGGGGCAGATTTCACTCTCACCATCAGCAG light CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGTTAAC chain AGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 258 413F09- Amino GFTFSYYA CDRH1 acid (IMGT) sequence of CDRH1 of 413F09 using IMGT 259 413F09- Amino ISGGGGNT CDRH2 acid (IMGT) sequence of CDRH2 of 413F09 using IMGT 260 413F09 Amino AKDRMKQLVRAYYFDY CDRH3 acid (IMGT) sequence of CDRH3 of 413F09 using IMGT 261 413F09 Amino YYAMS CDRH1 acid (Kabat) sequence of CDRH1 of 413F09 using Kabat 262 413F09- Amino TISGGGGNTHYADSVKG CDRH2 acid (Kabat) sequence of CDRH2 of 413F09 using Kabat 263 413F09- Amino DRMKQLVRAYYFDY CDRH3 acid (Kabat) sequence of CDRH3 of 413F09 using Kabat 264 413F09- Amino EVPLVESGGGLVQPGGSLRLSCAASGFTFSYYAMSWVRQAPGKGLD Heavy acid WVSTISGGGGNTHYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTA chain sequence VYYCAKDRMKQLVRAYYFDYWGQGTLVTVSS variable of region V_(H) of 413F09 265 413F09- Nucleic GAGGTGCCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGG Heavy acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCTA chain sequence CTATGCCATGAGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAC variable of TGGGTCTCAACTATTAGTGGTGGTGGTGGTAACACACACTACGCAG region V_(H) ACTCCGTGAAGGGCCGATTCACTATATCCAGAGACAATTCCAAGAA of CACGCTGTATCTGCACATGAACAGCCTGAGAGCCGAAGACACGGCC 413F09 GTCTATTACTGTGCGAAGGATCGGATGAAACAGCTCGTCCGGGCCT ACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC AG 266 413F09- Amino EVPLVESGGGLVQPGGSLRLSCAASGFTFSYYAMSWVRQAPGKGLD full acid WVSTISGGGGNTHYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTA heavy sequence VYYCAKDRMKQLVRAYYFDYWGQGTLVTVSSASTKGPSVFPLAPSS chain of KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG sequence 413F09 LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH heavy TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED chain PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 267 413F09- Nucleic GAGGTGCCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGG full acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCTA heavy sequence CTATGCCATGAGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAC chain of TGGGTCTCAACTATTAGTGGTGGTGGTGGTAACACACACTACGCAG sequence 413F09 ACTCCGTGAAGGGCCGATTCACTATATCCAGAGACAATTCCAAGAA heavy CACGCTGTATCTGCACATGAACAGCCTGAGAGCCGAAGACACGGCC chain GTCTATTACTGTGCGAAGGATCGGATGAAACAGCTCGTCCGGGCCT ACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC AGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGC AAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGG ACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCT GACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGC CTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGG GCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACAC CAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCAC ACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCG TGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCG GACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGAC CCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACA ACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCG GGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCA TCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCA GGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAG GTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCG CCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGAC CACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGC AAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCT CCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAA GTCCCTGTCCCTGAGCCCCGGCAAG 268 413F09- Amino QDISTY CDRL1 acid (IMGT) sequence of CDRL1 of 413F09 using IMGT 269 413F09- Amino GTS CDRL2 acid (IMGT) sequence of CDRL2 of 413F09 using IMGT 270 413F09- Amino QQLHTDPIT CDRL3 acid (IMGT) sequence of CDRL3 of 413F09 using IMGT 271 413F09- Amino WASQDISTYLG CDRL1 acid (Kabat) sequence of CDRL1 of 413F09 using Kabat 272 413F09- Amino GTSSLQS CDRL2 acid (Kabat) sequence of CDRL2 of 413F09 using Kabat 273 413F09- Amino QQLHTDPIT CDRL3 acid (Kabat) sequence of CDRL3 of 413F09 using Kabat 274 413F09 Amino DIQLTQSPSFLSASVGDRVTITCWASQDISTYLGWYQQKPGKAPKL Light acid LIYGTSSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLH chain sequence TDPITFGQGTRLEIK variable of region V_(L) of 413F09 275 413F09- Nucleic GACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAG Light acid GAGACAGAGTCACCATCACTTGCTGGGCCAGTCAGGACATTAGCAC chain sequence TTATTTAGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTC variable of CTGATCTATGGTACATCCAGTTTGCAAAGTGGGGTCCCATCAAGGT region V_(L) TCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAG of CCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTCAT 413F09 ACTGACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC 276 413F09- Amino DIQLTQSPSFLSASVGDRVTITCWASQDISTYLGWYQQKPGKAPKL full acid LIYGTSSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLH light sequence TDPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain of FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence 413F09 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 277 413F09- Nucleic GACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAG full acid GAGACAGAGTCACCATCACTTGCTGGGCCAGTCAGGACATTAGCAC light sequence TTATTTAGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTC chain of CTGATCTATGGTACATCCAGTTTGCAAAGTGGGGTCCCATCAAGGT sequence 413F09 TCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAG light CCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTCAT chain ACTGACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 278 414B06- Amino GFTFSSYW CDRH1 acid (IMGT) sequence of CDRH1 of 414B06 using IMGT 279 414B06- Amino IKQDGSEK CDRH2 acid (IMGT) sequence of CDRH2 of 414B06 using IMGT 280 414B06- Amino ARVRQWSDYSDY CDRH3 acid (IMGT) sequence of CDRH3 of 414B06 using IMGT 281 414B06- Amino SYWMN CDRH1 acid (Kabat) sequence of CDRH1 of 414B06 using Kabat 282 414B06- Amino NIKQDGSEKYYVDSVKG CDRH2 acid (Kabat) sequence of CDRH2 of 414B06 using Kabat 283 414B06- Amino VRQWSDYSDY CDRH3 acid (Kabat) sequence of CDRH3 of 414B06 using Kabat 284 414B06 Amino EVHLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLE Heavy acid WVANIKQDGSEKYYVDSVKGRFTVSRDNAKNSLYLQMNSLRAEDTA chain sequence VYYCARVRQWSDYSDYWGQGTPVTVSS variable of region V_(H) of 414B06 285 414B06- Nucleic GAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG Heavy acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAG chain sequence CTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG variable of TGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGG region V_(H) ACTCTGTGAAGGGCCGCTTCACCGTCTCCAGAGACAACGCCAAGAA of CTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCT 414B06 GTGTATTACTGTGCGAGAGTTCGACAATGGTCCGACTACTCTGACT ACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCAG 286 414B06- Amino EVHLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLE full acid WVANIKQDGSEKYYVDSVKGRFTVSRDNAKNSLYLQMNSLRAEDTA heavy sequence VYYCARVRQWSDYSDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTS chain of GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL sequence 414B06 SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP heavy CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK chain FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 287 414B06 Nucleic GAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGG full acid GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAG heavy sequence CTATTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAG chain of TGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGG sequence 414B06 ACTCTGTGAAGGGCCGCTTCACCGTCTCCAGAGACAACGCCAAGAA heavy CTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCT chain GTGTATTACTGTGCGAGAGTTCGACAATGGTCCGACTACTCTGACT ACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCAGCCAGCACCAA GGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCT GGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCG AGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGT GCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTG TCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCT ACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA GAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCT TGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCC CCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGT GACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCA AGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGT GCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAG TGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCA TCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACT GCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACC TGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGG AGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGT GCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTG GACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGA TGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCT GAGCCCCGGCAAG 288 414B06- Amino QGISSW CDRL1 acid (IMGT) sequence of CDRL1 of 414B06 using IMGT 289 414B06 Amino AAS CDRL2 acid (IMGT) sequence of CDRL2 of 414B06 using IMGT 290 414B06- Amino QQANSFPFT CDRL3 acid (IMGT) sequence of CDRL3 of 414B06 using IMGT 291 414B06- Amino RASQGISSWLA CDRL1 acid (Kabat) sequence of CDRL1 of 414B06 using Kabat 292 414B06- Amino AASSLQS CDRL2 acid 02 (Kabat) sequence of CDRL2 of 414B06 using Kabat 293 414B06- Amino QQANSFPFT CDRL3 acid (Kabat) sequence of CDRL3 of 414B06 using Kabat 294 414B06- Amino DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKL Light acid LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAN chain sequence SFPFTFGPGTKVDIK variable of region V_(L) of 414B06 295 414B06- Nucleic GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAG Light acid GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAG chain sequence CTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTC variable of CTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGT region V_(L) TCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG of CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAAC 414B06 AGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC 296 414B06- Amino DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKL full acid LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAN light sequence SFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN chain of FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA sequence 414B06 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC light chain 297 414B06- Nucleic GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAG full acid GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAG light sequence CTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTC chain of CTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGT sequence 414B06 TCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG light CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAAC chain AGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGA GCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAAC TTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCC TGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAA GGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGG GCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT 298 Mutated Amino DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL 1D05- acid LIY Y ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY LC sequence STPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN mutant of FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA 3 1D05 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC kappa light chain with V to Y mutation in CDRL2 highlighted 299 1D05 Amino EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLE heavy acid WVSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTA chain sequence LYYCAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPSS disabled of KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG IgG1 IgG1 LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH Fc disabled TCPPCPAPE LAGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED variant PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG of KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ 1D05 VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 300 1D05- 1D05 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKL light Light LIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSY chain chain STPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN IL-2 sequence FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA fusion fused DYEKHKVYACEVTHQGLSSPVTKSFNRGEC APTSSSTKKTQLQLEH to LLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEE wild-type LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA human DETATIVEFLNRWITFCQSIISTLT IL-2 sequence (IL- 2 amino acid sequence is underlined and region to be varied is shown in bold) 301 Human Uniprot APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM IL-2 number: PKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI P60568 VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT Full length amino acid sequence of human IL-2 (minus signal sequence) 302 Control Heavy EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLE 1D05 chain WVSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTA immunocy 1D05 LYYCAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPSS tokine IgG1 KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG HC variant LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH C- fused TCPPCPAPE LAGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED terminal at PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG fusion the KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ N-terminus VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS to KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAPTSSST wild-type KKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATEL human KHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGS IL2 ETTFMCEYADETATIVEFLNRWITFCQSIISTLT sequence (control) 303 IL-2 IL-2 APTSTQLQLELLLD D5- IC45 9 (Del 5- 9) N terminal IL-2 sequence 304 IL-2 IL-2 TQLQLEHLLLD D1- IC46 9 (Del 1- 9) N terminal IL-2 sequence 305 IL-2 IL-2 APTSKKTQLQLEHLLLD D5- IC64 7 (Del 5- 7) N terminal IL-2 sequence 306 IL-2 IL-2 PTSSSTKKTQLQLEHLLLD D1 D1 N terminal IL-2 sequence 307 IL-2 IL-2 TSSSTKKTQLQLEHLLLD D1- D1-2 2 N terminal IL-2 sequence 308 IL-2 IL-2 SSSTKKTQLQLEHLLLD D1- D1-3 3 N terminal IL-2 sequence 309 IL-2 IL-2 SSTKKTQLQLEHLLLD D1- D1-4 4 N terminal IL-2 sequence 310 IL-2 IL-2 STKKTQLQLEHLLLD D1- D1-5 5 N terminal IL-2 sequence 311 IL-2 IL-2 TKKTQLQLEHLLLD D1- D1-6 6 N terminal IL-2 sequence 312 IL-2 IL-2 KKTQLQLEHLLLD D1- D1-7 7 N terminal IL-2 sequence 313 IL-2 IL-2 KTQLQLEHLLLD D1- D1-8 8 N terminal IL-2 sequence 314 IL-2 IL-2 APTSSSTKTQLQLEHLLLD D9 D9 N terminal IL-2 sequence 315 IL-2 IL-2 APTSSSTTQLQLEHLLLD D9- D9-8 8 N terminal IL-2 sequence 316 IL-2 IL-2 APTSSSTQLQLEHLLLD D9- D9-7 7 N terminal IL-2 sequence 317 IL-2 IL-2 APTSSTQLQLEHLLLD D9- D9-6 6 N terminal IL-2 sequence 318 IL-2 IL-2 APTTQLQLEHLLLD D9- D9-4 4 N terminal IL-2 sequence 319 IL-2 IL-2 APTQLQLEHLLLD D9- D9-3 3 N terminal IL-2 sequence 320 IL-2 IL-2 ATQLQLEHLLLD D9- D9-2 2 N terminal IL-2 sequence 321 IL-2 IL-2 ATKKTQLQLEHLLLD D2- D2-6 6 N terminal IL-2 sequence 322 IL-2 IL-2 APKKTQLQLEHLLLD D3- D3-7 7 N terminal IL-2 sequence 323 IL-2 IL-2 APTKTQLQLEHLLLD D4- D4-8 8 N terminal IL-2 sequence 324 C- Amino LQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPL terminal acids EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETA amino 21 TIVEFLNRWITFCQSIISTLT acid to sequence 133 of of hIL-2 hIL-2 325 Mouse Uniprot MRIFAGIIFTACCHLLRAFTITAPKDLYVVEYGSNVTMECRFPVER PD-L1 number: ELDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQL Q9EP73 LKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKIN (ECD QRISVDPATSEHELICQAEGYPEAEVIWINSDHQPVSGKRSVTTSR highlighted TEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPA in THPPQNRT HWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVED BOLD, TSSKNRNDTQFEET and cytoplasmic domain underlined) 326 Mouse Mouse FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQV PD-L1 PD-L1 IQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAG ECD extracellular VYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQA His domain EGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATA with NDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRT HHHHHH his tag 327 Human Human ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLC IL-2RO IL-2 TGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPM chain receptor QPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRAL alpha HRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEG chain RPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLI SVLLLSGLTWQRRQRKSRRTI 328 Human Human AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQ IL-2Rß IL-2 TCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRV chain receptor MAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERH beta LEFEARTLS chain PGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRV KPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFG FIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQK WLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPAS LSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDE GVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSP PSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQ PPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLN TDAYLSLQELQGQDPTHLV 329 Human Human LNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVE IL-2RY IL-2 YMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSG chain receptor CQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENL common TLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRH gamma KFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSK chain ENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLED LVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGE GPGASPCNQHSPYWAPPCYTLKPET 330 IL-7 Human DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHI IL-7 CDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNC amino TGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEI acid KTCWNKILMGTKEH sequence 331 IL-15 Human GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYT IL-15 ESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNS amino LSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS acid sequence 332 IL-21 Human QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSA IL-21 FSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLT amino CPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS acid sequence 333 GM-CSF Human APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEM GM-CSF FDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTP amino ETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE acid sequence 334 IFNo Human CDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGS IFN-Q QLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTELHQQ amino LQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSD acid CAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGS sequence 335 TNFQ Extracellular GPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQL portion QWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPST of HVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPI human YLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL TNF-a amino acid sequence 336 IL-120 Alpha RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEE chain IDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASR of KTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNML human AVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRA IL-12 VTIDRVMSYLNAS amino acid sequence 337 IL-12ß Beta IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEV chain LGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWST of DILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR human GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESL IL-12 PIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQV amino EVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATV acid ICRKNASISVRAQDRYYSSSWSEWASVPCS sequence 338 CXCL9 Human TPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLK CXCL-9 NGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVR amino KSQRSRQKKTT acid sequence 339 CXCL10 Human VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMK CXCL-10 KKGEKRCLNPESKAIKNLLKAVSKERSKRSP amino acid sequence 340 Human IGHG1 WT human ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL WT *01 & IgG1 TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT IgG1 IGHG1 amino KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR constant *02 acid TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR region & sequence VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ IGHG1 VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT *05 TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK (IgG1) SLSLSPGK 341 WT human GCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCA IgG1 AGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGA nucleic CTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTG acid ACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCC sequence TGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGG CACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACC AAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACA CCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGT GTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGG ACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACC CTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAA CGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGG GTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCA AAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCAT CGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAG GTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGG TGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGC CGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACC ACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCA AGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTC CTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG TCCCTGTCCCTGAGCCCCGGCAAGTGATGA 342 Mutated Amino EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQ A PGKGLE 1D05- acid WVSGISWIRTGIGYADSVKGRFTI S RDNAKNSLYLQMNSLRAEDTA HC sequence LYYCAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCS mutant of RSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG 2 1D05 LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP heavy PCPAPE LAGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV chain QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY with KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL V to A TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT and VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK F to S back-mutation in framework region to germline highlighted with IgG1 disabled (LAGA) constant region

TABLE S2 SEQ ID NOS: 343-538 SEQ ID NO: Name Description Sequence 343 416E01- Amino acid GFTFSNYA CDRH1 sequence (IMGT) of CDRH1 of 416E01 using IMGT 344 416E01 Amino acid ISFSGGTT CDRH2 sequence (IMGT) of CDRH2 of 416E01 using IMGT 345 416E01- Amino acid AKDEAPAGATFFDS CDRH3 sequence (IMGT) of CDRH3 of 416E01 using IMGT 346 416E01 Amino acid NYAMS CDRH1 sequence (Kabat) of CDRH1 of 416E01 using Kabat 347 416E01- Amino acid AISFSGGTTYYADSVKG CDRH2 sequence (Kabat) of CDRH2 of 416E01 using Kabat 348 416E01 Amino acid DEAPAGATFFDS CDRH3 sequence (Kabat) of CDRH3 of 416E01 using Kabat 349 416E01- Amino acid EVQLAESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQTPGKGL Heavy sequence EWVSAISFSGGTTYYADSVKGRFTISRDNSKNTLYLHMNSLRADD chain of V_(H )of TAVYYCAKDEAPAGATFFDSWGQGTLVTVSS variable 416E01 region (mutations from germline are shown in bold letters) 350 416E01- Nucleic acid GAAGTGCAACTGGCGGAGTCTGGGGGAGGCTTGGTACAGCCGGGG Heavy sequence of GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC chain V_(H )of AACTATGCCATGAGTTGGGTCCGCCAGACTCCAGGAAAGGGGCTG variable 416E01 GAGTGGGTCTCAGCTATTAGTTTTAGTGGTGGTACTACATACTAC region GCTGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCC AAGAACACGCTGTATTTGCACATGAACAGCCTGAGAGCCGATGAC ACGGCCGTATATTACTGTGCGAAAGATGAGGCACCAGCTGGCGCA ACCTTCTTTGACTCCTGGGGCCAGGGAACGCTGGTCACCGTCTCC TCAG 351 416E01- Amino acid EVQLAESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQTPGKGL full sequence EWVSAISFSGGTTYYADSVKGRFTISRDNSKNTLYLHMNSLRADD heavy of 416E01 TAVYYCAKDEAPAGATFFDSWGQGTLVTVSSASTKGPSVFPLAPC chain heavy SRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS sequence chain SGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 352 416E01- Nucleic acid GAAGTGCAACTGGCGGAGTCTGGGGGAGGCTTGGTACAGCCGGGG full sequence of GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGC heavy 416E01 AACTATGCCATGAGTTGGGTCCGCCAGACTCCAGGAAAGGGGCTG chain heavy chain GAGTGGGTCTCAGCTATTAGTTTTAGTGGTGGTACTACATACTAC sequence GCTGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCC AAGAACACGCTGTATTTGCACATGAACAGCCTGAGAGCCGATGAC ACGGCCGTATATTACTGTGCGAAAGATGAGGCACCAGCTGGCGCA ACCTTCTTTGACTCCTGGGGCCAGGGAACGCTGGTCACCGTCTCC TCAGCCAGCACCAAGGGCCCTTCCGTGTTCCCCCTGGCCCCTTGC AGCAGGAGCACCTCCGAATCCACAGCTGCCCTGGGCTGTCTGGTG AAGGACTACTTTCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGC GCTCTGACATCCGGCGTCCACACCTTTCCTGCCGTCCTGCAGTCC TCCGGCCTCTACTCCCTGTCCTCCGTGGTGACCGTGCCTAGCTCC TCCCTCGGCACCAAGACCTACACCTGTAACGTGGACCACAAACCC TCCAACACCAAGGTGGACAAACGGGTCGAGAGCAAGTACGGCCCT CCCTGCCCTCCTTGTCCTGCCCCCGAGTTCGAAGGCGGACCCAGC GTGTTCCTGTTCCCTCCTAAGCCCAAGGACACCCTCATGATCAGC CGGACACCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCAGGAG GACCCTGAGGTCCAGTTCAACTGGTATGTGGATGGCGTGGAGGTG CACAACGCCAAGACAAAGCCCCGGGAAGAGCAGTTCAACTCCACC TACAGGGTGGTCAGCGTGCTGACCGTGCTGCATCAGGACTGGCTG AACGGCAAGGAGTACAAGTGCAAGGTCAGCAATAAGGGACTGCCC AGCAGCATCGAGAAGACCATCTCCAAGGCTAAAGGCCAGCCCCGG GAACCTCAGGTGTACACCCTGCCTCCCAGCCAGGAGGAGATGACC AAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCT TCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAAC AATTATAAGACCACCCCTCCCGTCCTCGACAGCGACGGATCCTTC TTTCTGTACTCCAGGCTGACCGTGGATAAGTCCAGGTGGCAGGAA GGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAAT CACTACACCCAGAAGTCCCTGAGCCTGTCCCTGGGAAAG 353 416E01- Amino acid QGIRRW CDRL1 sequence of CDRL1 (IMGT) of 416E01 using IMGT 354 416E01- Amino acid GAS CDRL2 sequence (IMGT) of CDRL2 of 416E01 using IMGT 355 416E01 Amino acid QQANSFPIT CDRL3 sequence (IMGT) of CDRL3 of 416E01 using IMGT 356 416E01- Amino acid RASQGIRRWLA CDRL1 sequence (Kabat) of CDRL1 of 416E01 using Kabat 357 416E01- Amino acid GASSLQS CDRL2 sequence (Kabat) of CDRL2 of 416E01 using Kabat 358 416E01- Amino acid QQANSFPIT CDRL3 sequence (Kabat) of CDRL3 of 416E01 using Kabat 359 416E01- Amino acid DIQMTQSPSSVSASVGDRVTITCRASQGIRRWLAWYQQKPGKAPK Light sequence LLISGASSLQSGVPSRFSGSGSGTDFTLIITSLQPEDFATYYCQQ chain of V_(L )of ANSFPITFGQGTRLEIK variable 416E01 region (mutations from germlin e are shown in bold letters) 360 416E01- Nucleic acid GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTA Light sequence of GGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGG chain V_(L )of AGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAA variable 416E01 CTCCTGATCTCTGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCA region AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCATCATT ACCAGTCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAG GCTAACAGTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAG ATCAAAC 361 416E01- Amino acid DIQMTQSPSSVSASVGDRVTITCRASQGIRRWLAWYQQKPGKAPK full sequence LLISGASSLQSGVPSRFSGSGSGTDFTLIITSLQPEDFATYYCQQ light of ANSFPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL chain 416E01 LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence light LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC chain 362 416E01- Nucleic GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTA full acid GGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGG light sequence of AGGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAA chain 416E01 CTCCTGATCTCTGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCA sequence light chain AGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCATCATT ACCAGTCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAG GCTAACAGTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAG ATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCT TCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTG CTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTG GACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAG CAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACC CTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAA GTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAAC CGGGGCGAGTGT 363 STIM001 Amino acid GYTFSTFG -CDRH1 sequence of CDRH1 of STIM001 using IMGT 364 STIM001 Amino acid ISAYNGDT -CDRH2 sequence of CDRH2 of STIM001 using IMGT 365 STIM001 Amino acid ARSSGHYYYYGMDV -CDRH3 sequence of CDRH3 of STIM001 using IMGT 366 STIM001 Amino acid QVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGL -Heavy sequence EWMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDD chain of V_(H )of TAVYYCARSSGHYYYYGMDVWGQGTTVTVSS variable STIM001 region 367 STIM001 Nucleic acid CAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGG -Heavy sequence of V_(H )of GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCC chain STIM001 ACCTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTT variable GAATGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACTAT region GCACAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCC ACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGAC ACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTAC TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCA 368 STIM001 Amino acid QVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGL -full sequence EWMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDD heavy of STIM001 heavy TAVYYCARSSGHYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPS chain chain SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS sequence SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K 369 STIM001 Nucleic acid CAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGG -full sequence of GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCC heavy STIM001 ACCTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTT chain heavy chain GAATGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACTAT sequence GCACAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCC ACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGAC ACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTAC TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC TCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCC AGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTG AAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGC GCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCC TCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGC TCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCC TCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGAC AAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGC GGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTG ATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTG TCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGC GTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTAC AACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAG GATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAG GCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGC CAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGAC GAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGC TTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAG CCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGAC GGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGG TGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCC CTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGC AAGTGATGA 370 STIM001 Amino acid QSLLHSNEYNY -CDRL1 sequence of CDRL1 of STIM001 using IMGT 371 STIM001 Amino acid LGS -CDRL2 sequence of CDRL2 of STIM001 using IMGT 372 STIM001 Amino acid MQSLQTPLT -CDRL3 sequence of CDRL3 of STIM001 using IMGT 373 STIM001 Amino acid DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKP -Light sequence GQSPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGI chain of V_(L )of YYCMQSLQTPLTFGGGTKVEIK variable STIM001 region 374 STIM001 Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCT -Light sequence of GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTG chain V_(L )of CATAGTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCA variable STIM001 GGGCAGTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCC region TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT TTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATT TATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAA 375 STIM001 Amino acid DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKP -full sequence GQSPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGI light of STIM001 light YYCMQSLQTPLTFGGGTKVEIK chain chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN sequence ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 376 STIM001 Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCT -full sequence of GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTG light STIM001 CATAGTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCA chain light chain GGGCAGTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCC sequence TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT TTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATT TATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAAcgtacggtggccgctccctccgtg ttcatcttcccaccttccgacgagcagctgaagtccggcaccgct tctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaag gtgcagtggaaggtggacaacgccctgcagtccggcaactcccag gaatccgtgaccgagcaggactccaaggacagcacctactccctg tcctccaccctgaccctgtccaaggccgactacgagaagcacaag gtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtg accaagtctttcaaccggggcgagtgt 377 STIM002 Amino acid GYTFTSYG -CDRH1 sequence of CDRH1 of STIM002 using IMGT 378 STIM002 Amino acid ISAYNGNT -CDRH2 sequence of CDRH2 of STIM002 using IMGT 379 STIM002 Amino acid ARSTYFYGSGTLYGMDV -CDRH3 sequence of CDRH3 of STIM002 using IMGT 380 STIM002 Amino acid QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGL Heavy sequence EWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDD chain of V_(H )of TAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSS variable STIM002 region 381 STIM002 Nucleic acid CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGG -Heavy sequence of V_(H )of GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACC chain STIM002 AGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTA variable GAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTAT region GCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCC ACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGAC ACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCG GGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCA 382 STIM002 Amino acid QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGL -full sequence EWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDD heavy of STIM002 heavy TAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSASTKGPSVFPL chain chain APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV sequence LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 383 STIM002 Nucleic acid CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGG -full sequence of GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACC heavy STIM002 AGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTA chain heavy chain GAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTAT sequence GCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCC ACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGAC ACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCG GGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCA GCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGC AAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAG GACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCT CTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCC GGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCT CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCC AACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAG ACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGA CCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATG ATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCC CACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTG GAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAAC TCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT TGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCC CTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAG CCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAG CTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTC TACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCT GAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGC TCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGG CAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG CACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG TGATGA 384 STIM002 Amino acid QSLLHSDGYNY -CDRL1 sequence of CDRL1 of STIM002 using IMGT 385 STIM002 Amino acid LGS -CDRL2 sequence of CDRL2 of STIM002 using IMGT 386 STIM002 Amino acid MQALQTPLS -CDRL3 sequence of CDRL3 of STIM002 using IMGT 387 STIM002 Amino acid DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKP -Light sequence GQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGV chain of V_(L )of YYCMQALQTPLSFGQGTKLEIK variable STIM002 region 388 STIM002 Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCT -Light sequence GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTG chain of CATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCA variable V_(L )of GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCC region STIM002 TCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT TTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTT TATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAG GGGACCAAGCTGGAGATCAAA 389 STIM002 Amino acid DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKP -full sequence GQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGV light of STIM002 YYCMQALQTPLSFGQGTKLEIK chain light RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN sequence chain ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 390 STIM002 Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCT -full sequence GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTG light of CATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCA chain STIM002 GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCC sequence light chain TCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT TTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTT TATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAG GGGACCAAGCTGGAGATCAAAcgtacggtggccgctccctccgtg ttcatcttcccaccttccgacgagcagctgaagtccggcaccgct tctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaag gtgcagtggaaggtggacaacgccctgcagtccggcaactcccag gaatccgtgaccgagcaggactccaaggacagcacctactccctg tcctccaccctgaccctgtccaaggccgactacgagaagcacaag gtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtg accaagtctttcaaccggggcgagtgt 391 STIM002- Amino acid GYTFTSYG B- sequence CDRH1 of CDRH1 of STIM002-B using IMGT 392 STIM002- Amino acid ISAYNGNT B- sequence CDRH2 of CDRH2 of STIM002-B using IMGT 393 STIM002- Amino acid ARSTYFYGSGTLYGMDV B- sequence CDRH3 of CDRH3 of STIM002-B using IMGT 394 STIM002- Amino acid QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGL B- sequence EWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDD Heavy of V_(H )of TAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSS chain STIM002-B variable region 395 STIM002- Nucleic acid CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGG B- sequence GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACC Heavy of AGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTA chain V_(H )of GAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTAT variable STIM002-B GCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCC region ACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGAC ACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCG GGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCA 396 STIM002- Amino acid QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGL B-full sequence EWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDD heavy of STIM002-B TAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSASTKGPSVFPL chain heavy APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV sequence chain LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 397 STIM002- Nucleic acid CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGG B-full sequence GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACC heavy of AGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTA chain STIM002-B GAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTAT sequence heavy GCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCC chain ACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGAC ACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCG GGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTG GCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGC TGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGG AACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTG CTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTG CCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAG TCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAA CTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAG GAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTG CTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAG GCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCT AGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTC GTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC AACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTG GACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGAC AAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG AGCCCCGGCAAGTGATGA 398 STIM002- Amino acid QSLLHSDGYNC B- sequence CDRL1 of CDRL1 of STIM002-B using IMGT 399 STIM002- Amino acid LGS B- sequence CDRL2 of CDRL2 of STIM002-B using IMGT 400 STIM002- Amino acid MQALQTPCS B- sequence CDRL3 of CDRL3 of STIM002-B using IMGT 401 STIM002- Amino acid DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKP B- sequence GQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGV Light of YYCMQALQTPCSFGQGTKLEIK chain V_(L )of variable STIM002-B region 402 STIM002- Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCT B- sequence GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTG Light of CATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCA chain V_(L )of GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCC variable STIM002-B TCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT region TTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTT TATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAG GGGACCAAGCTGGAGATCAAA 403 STIM002- Amino acid DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKP B-full sequence GQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGV light of STIM002-B YYCMQALQTPCSFGQGTKLEIK chain light RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN sequence chain ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 404 STIM002- Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCT B-full sequence GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTG light of CATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCA chain STIM002-B GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCC sequence light TCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT chain TTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTT TATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAG GGGACCAAGCTGGAGATCAAAcgtacggtggccgctccctccgtg ttcatcttcccaccttccgacgagcagctgaagtccggcaccgct tctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaag gtgcagtggaaggtggacaacgccctgcagtccggcaactcccag gaatccgtgaccgagcaggactccaaggacagcacctactccctg tcctccaccctgaccctgtccaaggccgactacgagaagcacaag gtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtg accaagtctttcaaccggggcgagtgt 405 STIM003 Amino acid GVTFDDYG -CDRH1 sequence of CDRH1 of STIM003 using IMGT 406 STIM003 Amino acid INWNGGDT -CDRH2 sequence of CDRH2 of STIM003 using IMGT 407 STIM003 Amino acid ARDFYGSGSYYHVPFDY -CDRH3 sequence of CDRH3 of STIM003 using IMGT 408 STIM003 Amino acid EVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGL -Heavy sequence EWVSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAED chain of V_(H )of TALYYCARDFYGSGSYYHVPFDYWGQGILVTVSS variable STIM003 region 409 STIM003 Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGG -Heavy sequence GGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGAT chain of V_(H )of GATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTG variable STIM003 GARTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTAT region TCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGAC ACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGT TATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTC ACCGTCTCCTCA 410 STIM003 Amino acid EVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGL 411 -full sequence EWVSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAED heavy of STIM003 TALYYCARDFYGSGSYYHVPFDYWGQGILVTVSSASTKGPSVFPL chain heavy APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV sequence chain LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK STIM003 Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGG -full sequence GGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGAT heavy of GATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTG chain STIM003 GARTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTAT sequence heavy chain TCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGAC ACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGT TATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTC ACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTG GCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGC TGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGG AACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTG CTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTG CCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAG TCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAA CTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAG GAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTG CTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAG GCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCT AGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTC GTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC AACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTG GACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGAC AAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG AGCCCCGGCAAGTGATGA 412 STIM003 Amino acid QSVSRSY -CDRL1 sequence of CDRL1 of STIM003 using IMGT 413 STIM003 Amino acid GAS -CDRL2 sequence of CDRL2 of STIM003 using IMGT 414 STIM003 Amino acid HQYDMSPFT -CDRL3 sequence of CDRL3 of STIM003 using IMGT 415 STIM003 Amino acid EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAP -Light sequence RLLIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCH chain of QYDMSPFTFGPGTKVDIK variable V_(L )of region STIM003 416 STIM003 Nucleic acid GAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCA -Light sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGC chain of AGAAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCC variable V_(L )of AGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA region STIM003 GACAGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCC ATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAC CAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTG GATATCAAA 417 STIM003 Amino acid EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAP -full sequence RLLIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCH light of STIM003 light QYDMSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVC chain chain LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL sequence TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 418 STIM003 Nucleic acid GAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCA -full sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGC light of AGAAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCC chain STIM003 AGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCA sequence light chain GACAGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCC ATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAC CAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTG GATATCAAAcgtacggtggccgctccctccgtgttcatcttccca ccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgc ctgctgaacaacttctacccccgcgaggccaaggtgcagtggaag gtggacaacgccctgcagtccggcaactcccaggaatccgtgacc gagcaggactccaaggacagcacctactccctgtcctccaccctg accctgtccaaggccgactacgagaagcacaaggtgtacgcctgc gaagtgacccaccagggcctgtctagccccgtgaccaagtctttc aaccggggcgagtgt 419 STIM004 Amino acid GLTFDDYG -CDRH1 sequence of CDRH1 of STIM004 using IMGT 420 STIM004 Amino acid INWNGDNT -CDRH2 sequence of CDRH2 of STIM004 using IMGT 421 STIM004 Amino acid ARDYYGSGSYYNVPFDY -CDRH3 sequence of CDRH3 of STIM004 using IMGT 422 STIM004 Amino acid EVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGL -Heavy sequence EWVSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAED chain of V_(H )of TALYYCARDYYGSGSYYNVPFDYWGQGTLVTVSS variable STIM004 region 423 STIM004 Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGG -Heavy sequence GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGAT chain of V_(H )of GATTATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTG variable STIM004 GAGTGGGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTAT region GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGAC ACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGT TATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCA 424 STIM004 Amino acid EVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGL -full sequence EWVSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAED heavy of STIM004 heavy TALYYCARDYYGSGSYYNVPFDYWGQGTLVTVSSASTKGPSVFPL chain chain APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV sequence LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 425 STIM004 Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGG -full sequence GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGAT heavy of GATTATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTG chain STIM004 GAGTGGGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTAT sequence heavy chain GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGAC ACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGT TATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTG GCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGC TGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGG AACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTG CTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTG CCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAG TCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAA CTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAG GAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTG CTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAG GCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCT AGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTC GTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC AACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTG GACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGAC AAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG AGCCCCGGCAAGTGATGA 426 STIM004 Amino acid QSVSSSY -CDRL1 sequence of CDRL1 of STIM004 using IMGT 427 STIM004 Amino acid GAS -CDRL2 sequence of CDRL2 of STIM004 using IMGT 428 STIM004 Amino acid QQYGSSPF -CDRL3 sequence of CDRL3 of STIM004 using IMGT 429 STIM004 Amino acid EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAP Corrected sequence RLLIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQ light of corrected QYGSSPFFGPGTKVDIK chain V_(L )of variable STIM004 region 430 STIM004- Nucleic acid GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCA Corrected sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGC light of AGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCC chain corrected AGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCA variable V_(L )of GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC region STIM004 ATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAG CAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGAT ATCAAA 431 STIM004 Nucleic acid GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCA -Light sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGC chain of AGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCC variable V_(L )of AGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCA region STIM004 GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC ATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAG CAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGG ATATCAAA 432 STIM004 Amino acid EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAP -full sequence RLLIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQ corrected of STIM004 light QYGSSPFFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCL light chain LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT chain LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC sequence 433 STIM004 Nucleic acid GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCA -full sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGC Corrected of AGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCC light corrected STIM004 AGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCA chain light chain GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC sequence ATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAG CAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGAT ATCAAAcgtacggtggccgctccctccgtgttcatcttcccacct tocgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctg ctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtg gacaacgccctgcagtceggcaactcccaggaatccgtgaccgag caggactccaaggacagcacctactccctgtcctccaccctgacc ctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaa gtgacccaccagggcctgtctagccccgtgaccaagtctttcaac cggggcgagtgt 434 STIM004 Nucleic acid GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCA -full sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGC light of AGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCC chain STIM004 AGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCA sequence light chain GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC ATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAG CAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGG ATATCAAAcgtacggtggccgctccctccgtgttcatcttcccac cttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcc tgctgaacaacttctacccccgcgaggccaaggtgcagtggaagg tggacaacgccctgcagtccggcaactcccaggaatccgtgaccg agcaggactccaaggacagcacctactccctgtcctccaccctga ccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcg aagtgacccaccagggcctgtctagccccgtgaccaagtctttca accggggcgagtgt 435 STIM005 Amino acid GYTFNSYG -CDRH1 sequence of CDRH1 of STIM005 using IMGT 436 STIM005 Amino acid ISVHNGNT -CDRH2 sequence of CDRH2 of STIM005 using IMGT 437 STIM005 Amino acid ARAGYDILTDFSDAFDI -CDRH3 sequence of CDRH3 of STIM005 using IMGT 438 STIM005 Amino acid QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGL -Heavy sequence EWMGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDD chain of V_(H )of TAVYYCARAGYDILTDFSDAFDIWGHGTMVTVSS variable STIM005 region 439 STIM005 Nucleic acid CAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGG -Heavy sequence GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAAT chain of V_(H )of AGTTATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTT variable STIM005 GAGTGGATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGT region GCACAGAAGCTCCAGGGTAGAGTCACCATGACCACAGACACATCC ACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGAC ACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACT GATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTC ACCGTCTCTTCA 440 STIM005 Amino acid QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGL -full sequence EWMGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDD heavy of STIM005 heavy TAVYYCARAGYDILTDFSDAFDIWGHGTMVTVSSASTKGPSVFPL chain chain APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV sequence LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 441 STIM005 Nucleic acid CAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGG -full sequence GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAAT heavy of AGTTATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTT chain STIM005 GAGTGGATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGT sequence heavy chain GCACAGAAGCTCCAGGGTAGAGTCACCATGACCACAGACACATCC ACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGAC ACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACT GATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTC ACCGTCTCTTCA GCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGC AAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAG GACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCT CTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCC GGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCT CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCC AACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAG ACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGA CCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATG ATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCC CACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTG GAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAAC TCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT TGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCC CTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAG CCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAG CTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTC TACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCT GAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGC TCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGG CAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG CACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG TGATGA 442 STIM005 Amino acid QNINNF -CDRL1 sequence of CDRL1 of STIM005 using IMGT 443 STIM005 Amino acid AAS -CDRL2 sequence of CDRL2 of STIM005 using IMGT 444 STIM005 Amino acid QQSYGIPW -CDRL3 sequence of CDRL3 of STIM005 using IMGT 445 STIM005 Amino acid DIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPK -Light sequence LLIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQ chain of V1 of SYGIPWVGQGTKVEIK variable STIM005 region 446 STIM005 Nucleic acid GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA -Light sequence GGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAAT chain of AACTTTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAG variable V_(L )of CTCCTGATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCA region STIM005 ACGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATC AGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAG AGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATC AAA 447 STIM005 Amino acid DIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPK -full sequence LLIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQ light of STIM005 light SYGIPWVGQGTKVEIK chain chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN sequence ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC 448 STIM005 Nucleic acid GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTA -full sequence GGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAAT light of AACTTTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAG chain STIM005 CTCCTGATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCA sequence light chain ACGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATC AGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAG AGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATC AAAcgtacggtggccgctccctccgtgttcatcttcccaccttcc gacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctg aacaacttctacccccgcgaggccaaggtgcagtggaaggtggac aacgccctgcagtceggcaactcccaggaatccgtgaccgagcag gactccaaggacagcacctactccctgtcctccaccctgaccctg tocaaggccgactacgagaagcacaaggtgtacgcctgcgaagtg acccaccagggcctgtctagccccgtgaccaagtctttcaaccgg ggcgagtgt 449 STIM006 Amino acid GFTFSDYF -CDRH1 sequence of CDRH1 of STIM006 using IMGT 450 STIM006 Amino acid ISSSGSTI -CDRH2 sequence of CDRH2 of STIM006 using IMGT 451 STIM006 Amino acid ARDHYDGSGIYPLYYYYGLDV -CDRH3 sequence of CDRH3 of STIM006 using IMGT 452 STIM006 Amino acid QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGL -Heavy sequence EWISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSED chain of V_(H )of TAVYYCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSS variable STIM006 region 453 STIM006 Nucleic acid CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGA -Heavy sequence GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT chain of V_(H )of GACTACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTG variable STIM006 GAGTGGATTTCATACATTAGTTCTAGTGGTAGTACCATATACTAC region GCAGACTCTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCC AAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGAC ACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGG ATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAG GGGACCACGGTCACCGTCTCCTCA 454 STIM006 Amino acid QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGL -full sequence EWISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSED heavy of STIM006 heavy TAVYYCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSSASTKGPS chain chain VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT sequence FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 455 STIM006 Nucleic acid CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGA -full sequence GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT heavy of GACTACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTG chain STIM006 GAGTGGATTTCATACATTAGTTCTAGTGGTAGTACCATATACTAC sequence heavy chain GCAGACTCTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCC AAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGAC ACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGG ATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAG GGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCT GTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACA GCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTG ACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACC TTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCC GTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATC TGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAG GTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGT CCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCC CCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG ACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACC AAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCC GTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTAC AAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAG ACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTAC ACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCC CTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTG GAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACC CCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAG CTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCC TGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG TCCCTGTCCCTGAGCCCCGGCAAGTGATGA 456 STIM006 Amino acid QSLLHSNGYNY -CDRL1 sequence of CDRL1 of STIM006 using IMGT 457 STIM006 Amino acid LGS -CDRL2 sequence of CDRL2 of STIM006 using IMGT 458 STIM006 Amino acid MQALQTPRS -CDRL3 sequence of CDRL3 of STIM006 using IMGT 459 STIM006 Amino acid IVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPG -Light sequence QSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVY chain of YCMQALQTPRSFGQGTTLEIK variable V_(L )of region STIM006 460 STIM006 Nucleic acid ATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGA -Light sequence GAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAT chain of AGTAATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGG variable V_(L )of CAGTCTCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCC region STIM006 GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTT ACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTAT TACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGG ACCACGCTGGAGATCAAA 461 STIM006 Amino acid IVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPG -full sequence QSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVY light of STIM006 YCMQALQTPRSFGQGTTLEIKRTVAAPSVFIFPPSDEQLKSGTAS chain light VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS sequence chain STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 462 STIM006 Nucleic acid ATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGA -full sequence GAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAT light of AGTAATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGG chain STIM006 CAGTCTCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCC sequence light chain GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTT ACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTAT TACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGG ACCACGCTGGAGATCAAAcgtacggtggccgctccctccgtgttc atcttcccaccttccgacgagcagctgaagtccggcaccgcttct gtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtg cagtggaaggtggacaacgccctgcagtccggcaactcccaggaa tccgtgaccgagcaggactccaaggacagcacctactccctgtcc tccaccctgaccctgtccaaggccgactacgagaagcacaaggtg tacgcctgcgaagtgacccaccagggcctgtctagccccgtgacc aagtctttcaaccggggcgagtgt 463 STIM007 Amino acid GFSLSTTGVG -CDRH1 sequence of CDRH1 of STIM007 using IMGT 464 STIM007 Amino acid IYWDDDK -CDRH2 sequence of CDRH2 of STIM007 using IMGT 465 STIM007 Amino acid THGYGSASYYHYGMDV -CDRH3 sequence of CDRH3 of STIM007 using IMGT 466 STIM007 Amino acid QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGK -Heavy sequence ALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPV chain of V_(H )of DTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSS variable STIM007 region 467 STIM007 Nucleic acid CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACA -Heavy sequence CAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGC chain of ACTACTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAG variable V_(H )of GCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGC region STIM007 TACAGCCCATCTCTGAAGAGCAGACTCACCATCACCAAGGACACC TCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTG GACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGT TATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCA 468 STIM007 Amino acid QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGK -full sequence ALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPV heavy of STIM007 heavy DTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSASTKGPSVFPL chain chain APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV sequence LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 469 STIM007 Nucleic acid CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACA -full sequence CAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGC heavy of ACTACTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAG chain STIM007 GCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGC sequence heavy chain TACAGCCCATCTCTGAAGAGCAGACTCACCATCACCAAGGACACC TCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTG GACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGT TATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCA GCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGC AAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAG GACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCT CTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCC GGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCT CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCC AACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAG ACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGA CCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATG ATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCC CACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTG GAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAAC TCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT TGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCC CTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAG CCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAG CTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTC TACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCT GAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGC TCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGG CAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTG CACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG TGATGA 470 STIM007- Amino acid QSVTNY CDRL1 sequence of CDRL1 of STIM007 using IMGT 471 STIM007- Amino acid DAS CDRL2 sequence of CDRL2 of STIM007 using IMGT 472 STIM007- Amino acid QHRSNWPLT CDRL3 sequence of CDRL3 of STIM007 using IMGT 473 STIM007 Amino acid EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPR -Light sequence LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQH chain of RSNWPLTFGGGTKVEIK variable V_(L )of region STIM007 474 STIM007 Nucleic acid GAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCA -Light sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACC chain of AACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGG variable V_(L )of CTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCC region STIM007 AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAC CGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAG ATCAAAC 475 STIM007 Amino acid EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPR -full sequence LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQH light of STIM007 light RSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL chain chain LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 476 STIM007 Nucleic acid GAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCA -full sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACC light of AACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGG chain STIM007 CTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCC sequence light chain AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAC CGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAG ATCAAACcgtacggtggccgctccctccgtgttcatcttcccacc ttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcct gotgaacaacttctacccccgcgaggccaaggtgcagtggaaggt ggacaacgccctgcagtccggcaactcccaggaatccgtgaccga gcaggactccaaggacagcacctactccctgtcctccaccctgac cctgtccaaggccgactacgagaagcacaaggtgtacgcctgcga agtgacccaccagggcctgtctagccccgtgaccaagtctttcaa ccggggcgagtgt 477 STIM008- Amino acid GFSLSTSGVG CDRH1 sequence of CDRH1 of STIM008 using IMGT 478 STIM008- Amino acid IYWDDDK CDRH2 sequence of CDRH2 of STIM008 using IMGT 479 STIM008- Amino acid THGYGSASYYHYGMDV CDRH3 sequence of CDRH3 of STIM008 using IMGT 480 STIM008 Amino acid QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGK -Heavy sequence ALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPV chain of V_(H )of DTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSS variable STIM008 region 481 STIM008 Nucleic acid CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACA -Heavy sequence CAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGC chain of V_(H )of ACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAG variable STIM008 GCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGC region TACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACC TCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTG GACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGT TATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCA 482 STIM008 Amino acid QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGK -full sequence ALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPV heavy of STIM008 heavy DTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSASTKGPSVFPL chain chain APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV sequence LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 483 STIM008 Nucleic acid CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACA -full sequence CAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGC heavy of ACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAG chain STIM008 GCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGC sequence heavy chain TACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACC TCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTG GACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGT TATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTC ACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTG GCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGC TGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGG AACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTG CTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTG CCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAAC CACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAG TCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAA CTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAG GACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTG GTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAG GAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTG CTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTG TCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAG GCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCT AGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTC GTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC AACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTG GACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGAC AAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATG CACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTG AGCCCCGGCAAGTGATGA 484 STIM008- Amino acid QSVTNY CDRL1 sequence of CDRL1 of STIM008 using IMGT 485 STIM008- Amino acid DAS CDRL2 sequence of CDRL2 of STIM008 using IMGT 486 STIM008- Amino acid QQRSNWPLT CDRL3 sequence of CDRL3 of STIM008 using IMGT 487 STIM008 Amino acid EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPR -Light sequence LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ chain of RSNWPLTFGGGTKVEIK variable V_(L )of region STIM008 488 STIM008 Nucleic acid GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCA -Light Sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACC chain of AACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGG variable V_(L )of CTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCC region STIM008 AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAG CGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAG ATCAAA 489 STIM008 Amino acid EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPR -full sequence LLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQ light of STIM008 light RSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL chain chain LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT sequence LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 490 STIM008 Nucleic acid GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCA -full sequence GGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACC light of AACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGG chain STIM008 CTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCC sequence light chain AGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATC AGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAG CGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAG ATCAAAcgtacggtggccgctccctccgtgttcatcttcccacct tccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctg ctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtg gacaacgccctgcagtceggcaactcccaggaatccgtgaccgag caggactccaaggacagcacctactccctgtcctccaccctgacc ctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaa gtgacccaccagggcctgtctagccccgtgaccaagtctttcaac cggggcgagtgt 491 STIM009- Amino acid GFTFSDYY CDRH1 sequence of CDRH1 of STIM009 using IMGT 492 STIM009- Amino acid ISSSGSTI CDRH2 sequence of CDRH2 of STIM009 using IMGT 493 STIM009- Amino acid ARDFYDILTDSPYFYYGVDV CDRH3 sequence of CDRH3 of STIM009 using IMGT 494 STIM009 Amino acid QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGL -Heavy sequence EWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAED chain of V_(H )of TAVYYCARDFYDILTDSPYFYYGVDVWGQGTTVTVSS variable STIM009 region 495 STIM009 Nucleic acid CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGA -Heavy sequence GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT chain of V_(H )of GACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTG variable STIM009 GAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTAC region GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCC AAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGAC ACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACT GATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGG ACCACGGTCACCGTCTCCTCA 496 STIM009 Amino acid QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGL -full sequence EWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAED heavy of STIM009 heavy TAVYYCARDFYDILTDSPYFYYGVDVWGQGTTVTVSSASTKGPSV chain chain FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF sequence PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 497 STIM009 Nucleic acid CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGA -full sequence GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT heavy of GACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTG chain STIM009 GAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTAC sequence heavy chain GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCC AAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGAC ACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACT GATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGG ACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTG TTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCC GCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACC GTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTC CCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTC GTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGC AACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTG GAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCT GCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCA AAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACC TGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTC AATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAG CCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTG CTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAG TGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACC ATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACA CTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTG ACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAA TGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCC CCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTG ACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGC TCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCC CTGTCCCTGAGCCCCGGCAAGTGATGA 498 STIM009- Amino acid QSLLHSNGYNY CDRL1 sequence of CDRL1 of STIM009 using IMGT 499 STIM009- Amino acid LGS CDRL2 sequence of CDRL2 of STIM009 using IMGT 500 STIM009- Amino acid MQALQTPRT CDRL3 sequence of CDRL3 of STIM009 using IMGT 501 STIM009 Amino acid DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP -Light sequence GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV chain of YYCMQALQTPRTFGQGTKVEIK variable V_(L )of region STIM009 502 STIM009 Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCT -Light sequence GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTG chain of CATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCA variable V_(L )of GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC region STIM009 TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT TTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTT TATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAA GGGACCAAGGTGGAAATCAAA 503 STIM009 Amino acid DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKP -full sequence GQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGV light of STIM009 light YYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTA chain chain SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL sequence SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 504 STIM009 Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCT -full sequence GGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTG light of CATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCA chain STIM009 GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCC sequence light chain TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT TTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTT TATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAA GGGACCAAGGTGGAAATCAAAcgtacggtggccgctccctccgtg ttcatcttcccaccttccgacgagcagctgaagtccggcaccgct tctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaag gtgcagtggaaggtggacaacgccctgcagtccggcaactcccag gaatccgtgaccgagcaggactccaaggacagcacctactccctg tcctccaccctgaccctgtccaaggccgactacgagaagcacaag gtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtg accaagtctttcaaccggggcgagtgt 505 Human Amino acid FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKN PD-L1 sequence IIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQD Flag His of KYPROT286 with AGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHEL (KYPROT2 FLAG tag in bold TCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLR 86) and underlined and INTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTIEGR histidine tag in DYKDDDDK HHHHHH bold 506 Mature Mature amino acid EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDL human sequence TKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCN ICOS of human LSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCI ICOS LGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL 507 Human Amino acid EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDL ICOS sequence TKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCN extra- of human ICOS LSIFDPPPFKVTLTGGYLHIYESQLCCQLKF cellular extracellular domain domain 508 Human Amino acid MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYP ICOS sequence DIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNN with of human ICOS SVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQL signal (signal peptide is CCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEY peptide underlined) MFMRAVNTAKKSRLTDVTL 509 Isoform Amino acid The sequence of human sequence of this isoform differs from the ICOS of a human ICOS canonical sequence in its cytoplasmic domain (Q9Y6W8- isoform as follows: 168-199: 2) KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLM 510 Mature Amino acid EINGSADHRMFSFHNGGVQISCKYPETVQQLKMRLFREREVLCEL mouse sequence TKTKGSGNAVSIKNPMLCLYHLSNNSVSFFLNNPDSSQGSYYFCS ICOS of mature mouse LSIFDPPPFQERNLSGGYLHIYESQLCCQLKIVVQVTE ICOS 511 Mouse Amino acid EINGSADHRMFSFHNGGVQISCKYPETVQQLKMRLFREREVLCEL ICOS sequence TKTKGSGNAVSIKNPMLCLYHLSNNSVSFFLNNPDSSQGSYYFCS extracellular of the LSIFDPPPFQERNLSGGYLHIYESQLCCQLK domain extracellular domain of mouse ICOS 512 Mouse Amino acid MGWSCIILFLVATATGVHSEINGSADHRMFSFHNGGVQISCKYPE ICOS sequence TVQQLKMRLFREREVLCELTKTKGSGNAVSIKNPMLCLYHLSNNS with of mouse ICOS VSFFLNNPDSSQGSYYFCSLSIFDPPPFQERNLSGGYLHIYESQL signal (signal peptide is CCQLKIVVQVTE peptide underlined) 513 Cynomolgus Amino acid MKSGLWYFFL FCLHMKVLTG EINGSANYEM FIFHNGGVQI ICOS sequence LCKYPDIVQQ with of cynomolgus ICOS FKMQLLKGGQILCDLTKTKGSGNKVSIKSLKFCHSQLSNNSVSFF signal (signal peptide is LYNLD peptide underlined) RSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPI GCATF VVVCIFGCILICWLTKKKYSSTVHDPNGEYMFMRAVNTAKKSRLT GTTP 514 Cynomolgus Amino acid EINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDL ICOS sequence TKTKG extracellular of cynomolgus ICOS SGNKVSIKSLKFCHSQLSNNSVSFFLYNLDRSHANYYFCNLSIFD domain extracellular PPPFKVTLTGGYLHIYESQLCCQLK domain 515 Human Amino acid DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVV ICOS sequence TYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDE ligand of human ICOS QKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDE ligand comprising LTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYD extracellular VVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKIT domain ENPVSTGEKNAATWS 516 Human Amino acid MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRF ICOS sequence DLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAG ligand of human ICOS MLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVA ligand including ANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLL signal peptide DQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQN LTVGSQTGNDIGERDKITENPVSTGEKNAATWSILAVLCLLVVVA VAIGWVCRDRCLQHSYAGAWAVSPETELTGHV 517 C-terminal amino Amino acids 21 to LQMILNGINNYKNPKLT A MLTFKFYMPKKATELKHLQC acid sequence 133 of hIL-2 with LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL of R38W mutation KGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT hIL-2 (bold & underlined) 518 C-terminal amino Amino acids 21 to LQMILNGINNYKNPKLT Q MLTFKFYMPKKATELKHLQC acid sequence 133 of hIL-2 with LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL of R38Q mutation KGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT hIL-2 (bold & underlined) 519 STIM002- Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGT Corrected Light sequence CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTA chain variable of GTCAGAGCCTCCTGCATAGTGATGGATACAACTATTTG region corrected V_(L )of GATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCT STIM002 CCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCC CTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTT ACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGG GGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCA GTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 520 STIM002 Nucleic acid GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGT Corrected sequence CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTA full of GTCAGAGCCTCCTGCATAGTGATGGATACAACTATTTG light corrected GATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCT chain STIM002 CCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCC sequence light chain CTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTT ACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGG GGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCA GTTTTGGCCAGGGGACCAAGCTGGAGATCAAAcgtacg gtggccgctccctccgtgttcatcttcccaccttccga cgagcagctgaagtccggcaccgcttctgtcgtgtgcc tgctgaacaacttctacccccgcgaggccaaggtgcag tggaaggtggacaacgccctgcagtccggcaactccca ggaatccgtgaccgagcaggactccaaggacagcacct actccctgtcctccaccctgaccctgtccaaggccgac tacgagaagcacaaggtgtacgcctgcgaagtgaccca ccagggcctgtctagccccgtgaccaagtctttcaacc ggggcgagtgt 521 STIM003- Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACG Corrected heavy Sequence GCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTG chain variable of GAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGC region corrected VH CAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTAT of STIM003 TAATTGGAATGGTGGCGACACAGATTATTCAGACTCTG TGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAG AACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGA GGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATG GTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGG GGCCAGGGAATCCTGGTCACCGTCTCCTCA 522 STIM003- Nucleic acid GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACG Corrected full sequence GCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTG heavy chain of GAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGC sequence corrected CAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTAT STIM003 TAATTGGAATGGTGGCGACACAGATTATTCAGACTCTG heavy TGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAG chain AACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGA GGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATG GTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGG GGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCAGCAC CAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCA AGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTC GTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTG GAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCC CTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCC TCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCA GACCTACATCTGCAACGTGAACCACAAGCCCTCCAACA CCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGAC AAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACT GCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGC CCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG ACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGA AGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGC ACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAAC TCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCA CCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGG TGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACC ATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGT GTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGA ACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTAC CCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCA GCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGG ACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACA GTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTC CTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACA CCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA 523 Human IGHG Human gcctccaccaagggcccatcggtcttccccctggcacc IgG1 1*03 Heavy ctcctccaagagcacctctgggggcacagcggccctgg constant Chain gctgcctggtcaaggactacttccccgaaccggtgacg region Constant gtgtcgtggaactcaggcgccctgaccagcggcgtgca Region caccttcccggctgtcctacagtcctcaggactctact (IGHG1*03) ccctcagcagcgtggtgaccgtgccctccagcagcttg Nucleotide ggcacccagacctacatctgcaacgtgaatcacaagcc Sequence cagcaacaccaaggtggacaagagagttgagcccaaat cttgtgacaaaactcacacatgcccaccgtgcccagca cctgaactcctggggggaccgtcagtcttcctcttccc cccaaaacccaaggacaccctcatgatctcccggaccc ctgaggtcacatgcgtggtggtggacgtgagccacgaa gaccctgaggtcaagttcaactggtacgtggacggcgt ggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcacc gtcctgcaccaggactggctgaatggcaaggagtacaa gtgcaaggtctccaacaaagccctcccagcccccatcg agaaaaccatctccaaagccaaagggcagccccgagaa ccacaggtgtacaccctgcccccatcccgggaggagat gaccaagaaccaggtcagcctgacctgcctggtcaaag gcttctatcccagcgacatcgccgtggagtgggagagc aatgggcagccggagaacaactacaagaccacgcctcc cgtgctggactccgacggctccttcttcctctatagca agctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaa ccactacacgcagaagagcctctccctgtccccgggta aa 524 Human A S T K G P S V F P L A P S S K S T S Heavy G G T A A L G C L V K D Y F P E P V T Chain V S W N S G A L T S G V H T F P A V L  Constant Q S S G L Y S L S S V V T V P S S S L Region G T Q T Y I C N V N H K P S N T K V D (IGHG1*03) K R V E P K S C D K T H T C P P C P A Protein P E L L G G P S V F L F P P K P K D T Sequence L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R E E M T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K 525 Human IGHG Human Heavy gcctccaccaagggcccatcggtcttccccctggcacc IgG1 1*04 Chain ctcctccaagagcacctctgggggcacagcggccctgg constant Constant gctgcctggtcaaggactacttccccgaaccggtgacg region Region gtgtcgtggaactcaggcgccctgaccagcggcgtgca (IGHG1*04) caccttcccggctgtcctacagtcctcaggactctact Nucleotide ccctcagcagcgtggtgaccgtgccctccagcagcttg Sequence ggcacccagacctacatctgcaacgtgaatcacaagcc cagcaacaccaaggtggacaagaaagttgagcccaaat cttgtgacaaaactcacacatgcccaccgtgcccagca cctgaactcctggggggaccgtcagtcttcctcttccc cccaaaacccaaggacaccctcatgatctcccggaccc ctgaggtcacatgcgtggtggtggacgtgagccacgaa gaccctgaggtcaagttcaactggtacgtggacggcgt ggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgtgtggtcagcgtcctcacc gtcctgcaccaggactggctgaatggcaaggagtacaa gtgcaaggtctccaacaaagccctcccagcccccatcg agaaaaccatctccaaagccaaagggcagccccgagaa ccacaggtgtacaccctgcccccatcccgggatgagct gaccaagaaccaggtcagcctgacctgcctggtcaaag gcttctatcccagcgacatcgccgtggagtgggagagc aatgggcagccggagaacaactacaagaccacgcctcc cgtgctggactccgacggctccttcttcctctacagca agctcaccgtggacaagagcaggtggcagcaggggaac atcttctcatgctccgtgatgcatgaggctctgcacaa ccactacacgcagaagagcctctccctgtctccgggta aa 526 Human Heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT Chain VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL Constant Region GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA (IGHG1*04) PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE Protein DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT Sequence VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN IFSCSVMHEALHNHYTQKSLSLSPGK 527 Human IGHG Human Heavy gcctccaccaagggcccatcggtcttccccctggcgcc IgG2 2*01 Chain ctgctccaggagcacctccgagagcacagccgccctgg constant & Constant gctgcctggtcaaggactacttccccgaaccggtgacg region IGHG Region gtgtcgtggaactcaggcgctctgaccagcggcgtgca 2*03 (IGHG2*01) caccttcccagctgtcctacagtcctcaggactctact & Nucleotide ccctcagcagcgtggtgaccgtgccctccagcaacttc IGHG Sequence ggcacccagacctacacctgcaacgtagatcacaagcc 2*05 cagcaacaccaaggtggacaagacagttgagcgcaaat gttgtgtcgagtgcccaccgtgcccagcaccacctgtg gcaggaccgtcagtcttcctcttccccccaaaacccaa ggacaccctcatgatctcccggacccctgaggtcacgt gcgtggtggtggacgtgagccacgaagaccccgaggtc cagttcaactggtacgtggacggcgtggaggtgcataa tgccaagacaaagccacgggaggagcagttcaacagca cgttccgtgtggtcagcgtcctcaccgttgtgcaccag gactggctgaacggcaaggagtacaagtgcaaggtctc caacaaaggcctcccagcccccatcgagaaaaccatct ccaaaaccaaagggcagccccgagaaccacaggtgtac accctgcccccatcccgggaggagatgaccaagaacca ggtcagcctgacctgcctggtcaaaggcttctacccca gcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacacctcccatgctggactc cgacggctccttcttcctctacagcaagctcaccgtgg acaagagcaggtggcagcaggggaacgtcttctcatgc tocgtgatgcatgaggctctgcacaaccactacacgca gaagagcctctccctgtctccgggtaaa 528 Human Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT Constant Region VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNF (IGHG2*01) GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPV Protein Sequence AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 529 Human IGHG Human Heavy Chain GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCC IgG2 2*02 Constant Region CTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGG constant (IGHG2*02) GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG region Nucleotide GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCA Sequence CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT CCCTCAGCAGCGTGGTGACCGTGACCTCCAGCAACTTC GGCACCCAGACCTACACCTGCAACGTAGATCACAAGCC CAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAAT GTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTG GCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGT GCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTC CAGTTCAACTGGTACGTGGACGGCATGGAGGTGCATAA TGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCA CGTTCCGTGTGGTCAGCGTCCTCACCGTCGTGCACCAG GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTC CAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCT CCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACACCTCCCATGCTGGACTC CGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACACA GAAGAGCCTCTCCCTGTCTCCGGGTAAA 530 Human Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT Constant Region VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNF (IGHG2*02) GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPV Protein Sequence AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 531 Human IGHG Human Heavy gcctccaccaagggcccatcggtcttccccctggcgcc IgG2 2*04 Chain Region ctgctccaggagcacctccgagagcacagcggccctgg constant Constant gctgcctggtcaaggactacttccccgaaccggtgacg region (IGHG2*04) gtgtcgtggaactcaggcgctctgaccagcggcgtgca Nucleotide caccttcccagctgtcctacagtcctcaggactctact Sequence ccctcagcagcgtggtgaccgtgccctccagcagcttg ggcacccagacctacacctgcaacgtagatcacaagcc cagcaacaccaaggtggacaagacagttgagcgcaaat gttgtgtcgagtgcccaccgtgcccagcaccacctgtg gcaggaccgtcagtcttcctcttccccccaaaacccaa ggacaccctcatgatctcccggacccctgaggtcacgt gcgtggtggtggacgtgagccacgaagaccccgaggtc cagttcaactggtacgtggacggcgtggaggtgcataa tgccaagacaaagccacgggaggagcagttcaacagca cgttccgtgtggtcagcgtcctcaccgttgtgcaccag gactggctgaacggcaaggagtacaagtgcaaggtctc caacaaaggcctcccagcccccatcgagaaaaccatct ccaaaaccaaagggcagccccgagaaccacaggtgtac accctgcccccatcccgggaggagatgaccaagaacca ggtcagcctgacctgcctggtcaaaggcttctacccca gcgacatcgccgtggagtgggagagcaatgggcagccg gagaacaactacaagaccacacctcccatgctggactc cgacggctccttcttcctctacagcaagctcaccgtgg acaagagcaggtggcagcaggggaacgtcttctcatgc tccgtgatgcatgaggctctgcacaaccactacacgca gaagagcctctccctgtctccgggtaaa 532 Human Heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT Chain VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL Constant GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPV Region AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV (IGHG2*04) QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ Protein DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVY Sequence TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 533 Human IGHG Human Heavy GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCC IgG2 2*06 Chain CTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGG constant Constant GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG region Region GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCA (IGHG2*06) CACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT Nucleotide CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTC Sequence GGCACCCAGACCTACACCTGCAACGTAGATCACAAGCC CAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAAT GTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTG GCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGT GCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTC CAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA TGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCA CGTTCCGTGTGGTCAGCGTCCTCACCGTCGTGCACCAG GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTC CAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCT CCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCA GCGACATCTCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACACCTCCCATGCTGGACTC CGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACACA GAAGAGCCTCTCCCTGTCTCCGGGTAAA 534 Human Heavy Chain ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT Constant Region VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNF (IGHG2*06) GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPV Protein Sequence AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQP ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 535 Human CA IGLC Cλ Light Chain GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCC constant 7*03 Constant Region ACCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACAC region (IGLC7*03) TGGTGTGTCTCGTAAGTGACTTCAACCCGGGAGCCGTG Nucleotide ACAGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGT Sequence GGGAGTGGAGACCACCAAACCCTCCAAACAAAGCAACA ACAAGTATGCGGCCAGCAGCTACCTGAGCCTGACGCCC GAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCGGGT CACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCC CTGCAGAATGCTCT 536 Cλ Light Chain GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFNPGAV Constant Region TVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTP (IGLC7*03) Amino EQWKSHRSYSCRVTHEGSTVEKTVAPAECS Acid Sequence 537 Human WT IGHG WT human IgG1 gcctccaccaagggcccatcggtcttccccctggcacc IgG1 1*01 nucleotide ctcctccaagagcacctctgggggcacagcggccctgg constant & sequence #2 gctgcctggtcaaggactacttccccgaaccggtgacg region IGHG gtgtcgtggaactcaggcgccctgaccagcggcgtgca 1*05 caccttcccggctgtcctacagtcctcaggactctact (IgG ccctcagcagcgtggtgaccgtgccctccagcagcttg 1) ggcacccagacctacatctgcaacgtgaatcacaagcc cagcaacaccaaggtggacaagaaagttgagcccaaat cttgtgacaaaactcacacatgcccaccgtgcccagca cctgaactcctggggggaccgtcagtcttcctcttccc cccaaaacccaaggacaccctcatgatctcccggaccc ctgaggtcacatgcgtggtggtggacgtgagccacgaa gaccctgaggtcaagttcaactggtacgtggacggcgt ggaggtgcataatgccaagacaaagccgcgggaggagc agtacaacagcacgtaccgggtggtcagcgtcctcacc gtcctgcaccaggactggctgaatggcaaggagtacaa gtgcaaggtctccaacaaagccctcccagcccccatcg agaaaaccatctccaaagccaaagggcagccccgagaa ccacaggtgtacaccctgcccccatcccgggatgagct gaccaagaaccaggtcagcctgacctgcctggtcaaag gcttctatcccagcgacatcgccgtggagtgggagagc aatgggcagccggagaacaactacaagaccacgcctcc cgtgctggactccgacggctccttcttcctctacagca agctcaccgtggacaagagcaggtggcagcaggggaac gtcttctcatgctccgtgatgcatgaggctctgcacaa ccactacacgcagaagagcctctccctgtctccgggta aa 538 Human CA IGLC Cλ Light Chain GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV constant 2*01 Constant Region TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTP region Amino Acid EQWKSHRSYSCQVTHEGSTVEKTVAPTECS Sequence #2- Encoded by nucleotide sequence version A & B

TABLE S3 SEQ ID NOS: 539-562 Sequence hIgG1 FIT-Ig bispecific 1a Antibody A anti-ICOS STIM003 Antibody B anti-PD-L1 84G09 FIT-Ig SEQ ID NO: 539 DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #1 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFI RSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHN TFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK FIT-Ig SEQ ID NO: 540  EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISY Construct #2 EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWED WGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKV FIT-Ig SEQ ID NO: 541 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #3 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGD GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC hIgG1 FIT-Ig bispecific 1b Antibody A anti-PD-L1 84G09 Antibody B anti-ICOS STIM003 FIT-Ig SEQ ID NO: 542 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #1 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGD GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGL EWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCAR QREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FIT-Ig SEQ ID NO: 543 EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRS Construct #2 GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTF DSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKV FIT-Ig SEQ ID NO: 544 DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #3 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC hIgG1 FIT-Ig bispecific 2a Antibody A anti-ICOS STIM001 Antibody B anti-PD-L1 1D05 FIT-Ig SEQ ID NO: 545 DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #1 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEI KRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGV LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN ECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFI RSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHN TFDSWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVT LTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWM SGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVER NSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO: 546 EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISY Construct #2 EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWED WGQGVMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK KI FIT-Ig SEQ ID NO: 547 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #3 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGD GTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGS ERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEC hIgG1 FIT-Ig bispecific 2b Antibody A anti-PD-L1 1D05 Antibody B anti-ICOS STIM001 FIT-Ig SEQ ID NO: 548 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #1 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGD GTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGS ERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGL EWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCAR QREANWEDWGQGVMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFP EPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHP ASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSP IVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQH QDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQV TLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKN WVERNSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO: 549 EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRS Construct #2 GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTF DSWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLT WNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKV DKKI FIT-Ig SEQ ID NO: 550 DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #3 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEI KRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGV LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN EC hIgG1 FIT-Ig bispecific 3a Antibody A anti-ICOS STIM003 Antibody B anti-PD-L1 1D05 FIT-Ig SEQ ID NO: 551 DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #1 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFI RSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHN TFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKE FKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTD FMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO: 552 EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISY Construct #2 EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWED WGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKV FIT-Ig SEQ ID NO: 553 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #3 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGD GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC hIgG1 FIT-Ig bispecific 3b Antibody A anti-PD-L1 1D05 Antibody B anti-ICOS STIM003 FIT-Ig SEQ ID NO: 554 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #1 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGD GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGL EWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCAR QREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWM SGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVER NSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO: 555 EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRS Construct #2 GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTF DSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKV FIT-Ig SEQ ID NO: 556 DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #3 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC hIgG1 FIT-Ig bispecific 4a Antibody A anti-ICOS STIM001 Antibody B anti-PD-L1 84G09 FIT-Ig SEQ ID NO: 557 DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #1 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFI RSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHN TFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKE FKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTD FMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYS CSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO: 558 EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISY Construct #2 EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWED WGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKV FIT-Ig SEQ ID NO: 559 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #3 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGD GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC hIgG1 FIT-Ig bispecific 4b Antibody A anti-PD-L1 84G09 Antibody B anti-ICOS STIM001 FIT-Ig SEQ ID NO: 560 DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLL Construct #1 IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGD GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGL EWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCAR QREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWM SGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVER NSYSCSVVHEGLHNHHTTKSFSRTPGK FIT-Ig SEQ ID NO: 561 EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRS Construct #2 GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTF DSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKV FIT-Ig SEQ ID NO: 562 DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASS Construct #3 LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC motif X₁GSGX₂YGX₃X₄FD (SEQ ID NO: 609) SEQ ID NO: 610 ICOSL-Fc DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLF NVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQAL QNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWS DIEGRMD PK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Linker is underlined and in bold. Sequence preceding linker is human ICOSL (B7-H2). Sequence following linker is human IgG1 Fc.

TABLE S4 Sequences of anti-ICOS antibody heavy chain variable regions obtained from additional clones CDRs are defined according to IMGT. VH_AMINO_ CLONE_ID VH_NUCLEOTIDE_SEQUENCE ACID_SEQ HCDR1 HCDR2 HCDR3 CL-61091 CAGGTTCAACTGATGCAGTCTGGAACT QVQLMQSGTE GYTFTTYG ISAYSGDT ARSSGWPHHYGMDV GAGGTGAAGAAGCCTGGGGCCTCAGTG VKKPGASVKV SEQ ID SEQ ID SEQ ID NO: 567 AAGGTCTCCTGCAAGACTTCTGGTTAC SCKTSGYTFT NO: 565 NO: 566 ACCTTTACCACCTATGGTATCACTTGG TYGITWVRçA GTGCGACAGGCCCCTGGACAAGGGCTT PGQGLEWMGW GAGTGGATGGGATGGATCAGCGCTTAC ISAYSGDTDY AGTGGTGACACAGACTATGCACAGAAG AQKFQGRVTV TTCCAGGGCAGAGTCACCGTGACAACA TTDTSTNTAY GACACATCCACGAACACAGCCTACATG MELRSLKSDD GAGTTGAGGAGCCTGAAATCTGACGAC TAVYYCARSS ACGGCCGTGTATTATTGTGCGAGAAGT GWPHHYGMDV AGTGGCTGGCCCCACCACTACGGTATG WGQGTTVTVS GACGTCTGGGGCCAAGGGACCACGGTC S ACCGTCTCCTCAG SEQ ID NO: SEQ ID NO: 563 564 CL-64536 CAGGTTCAACTGGTGCAGTCTGGAGGT QVOLVOSGGE GYTFTSYG ISAYNGNT ARSTSYYGSGTLYG GAGGTGAAAAAGCCTGGGGCCTCAGTG VKKPGASVKV SEQ ID SEQ ID MDV AAGGTCTCCTGCAAGGCTTCTGGTTAC SCKASGYTFT NO: 377 NO: 378 SEQ ID NO: 570 ACCTTTACCAGCTATGGTTTCAGCTGG SYGFSWVRQA GTGCGACAGGCCCCTGGACAAGGACTA PGQGLEWMGW GAGTGGATGGGATGGATCAGCGCTTAC ISAYNGNTNY AATGGTAACACAAACTATGCACAGAAG AQKLQGRVSM CTCCAGGGCAGAGTCTCCATGACCACA TTDTSTSTAY GACACATCCACGAGCACAGCCTACATG MELRSLRSDD GAGCTGAGGAGCTTGAGATCTGACGAC TAVYFCARST ACGGCCGTGTATTTCTGTGCGCGATCT SYYGSGTLYG ACGTCTTACTATGGTTCGGGGACCCTA MDVWGQGTTV TACGGTATGGACGTCTGGGGCCAAGGG TVSS ACCACGGTCACCGTCTCCTCAG SEQ ID NO: SEQ ID NO: 568 569 CL-64837 CAGGTTCAACTGGTGCAGTCTGGAGGT QVQLVQSGGE GYTFTSYG ISAYNGNT ARSTSYYGSGTLYG GAGGTGAAGAAGCCTGGGGCCTCAGTG VKKPGASVKV SEQ ID SEQ ID MDV AAGGTCTCCTGCAAGGCTTCTGGTTAC SCKASGYTFT NO: 377 NO: 378 SEQ ID NO: 570 ACCTTTACCAGCTATGGTTTCAGCTGG SYGFSWVRQA GTGCGACAGGCCCCTGGACAAGGACTA PGQGLEWMGW GAGTGGATGGGATGGATCAGCGCTTAC ISAYNGNTNY AATGGTAACACAAACTATGCACAGAAG AQKLQGRVSM CTCCAGGGCAGAGTCTCCATGACCACA TTDTSTSTAY GACACATCCACGAGCACAGCCTACATG MELRSLRSDD GAGCTGAGGAGCTTGAGATCTGACGAC TAVYYCARST ACGGCCGTGTATTACTGTGCGCGATCT SYYGSGTLYG ACGTCTTACTATGGTTCGGGGACCCTC MDVWGQGTTV TACGGTATGGACGTCTGGGGCCAAGGG TVSS ACCACGGTCACCGTCTCCTCAG SEQ ID NO: SEQ ID NO: 571 572 CL-64841 CAGGTTCAACTGGTGCAGTCTGGAGGT QVQLVQSGGE GYTFTSYG ISAYNGNT ARSTSYYGSGTLYG GAGGTGAAAAAGCCTGGGGCCTCAGTG VKKPGASVKV SEQ ID SEQ ID MDV AAGGTCTCCTGCAAGGCTTCTGGTTAC SCKASGYTFT NO: 377 NO: 378 SEQ ID NO: 570 ACCTTTACCAGCTATGGTTTCAGCTGG SYGFSWVRçA GTGCGACAGGCCCCTGGACAAGGACTA PGQGLEWMGW GAGTGGATGGGATGGATCAGCGCTTAC ISAYNGNTNY AATGGTAACACAAACTATGCACAGAAG AQKLQGRVSM CTCCAGGGCAGAGTCTCCATGACCACA TTDTSTSTAY GACACATCCACGAGCACAGCCTACATG MELRSLRSDD GAGCTGAGGAGCTTGAGATCTGACGAC TAVYFCARST ACGGCCGTGTATTTCTGTGCGCGATCT SYYGSGTLYG ACGTCTTACTATGGTTCGGGGACCCTA MDVWGQGTTV TACGGTATGGACGTCTGGGGCCAAGGG TVSS ACCACGGTCACCGTCTCCTCAG SEQ ID NO: SEQ ID NO: 573 574 CL-64912 CAGGTTCAACTGGTGCAGTCTGGAGGT QVQLVQSGGE GYTFTSYV ISGYNGNT ARSTSYYGAGTLYG GAGGTGAAAAAGCCTCGGGCCTCAGTG VKKPRASVKV SEQ ID SEQ ID MDV AAGGTCTCCTGCAAGGCTTCTGGTTAC SCKASGYTFT NO: 577 NO: 578 SEQ ID NO: 579 ACCTTTACCAGCTATGTGTTCAGCTGG SYVFSWVRHA GTGCGACATGCCGCTGGACAAGGACTA AGQGLEWMGW GAGTGGATGGGATGGATCAGCGGTTAC ISGYNGNTNY AATGGTAACACAAACTATGCACAGAAG AQKLQCGVSM CTCCAGTGCGGAGTCTCGATGACCGCA TADTSTSTAY GACACATCCACGAGCACAGCCTACATG MELRSLRSDD GAGCTGAGGAGCTTGAGATCTGACGAC TAVYFCARST ACGGCCGTGTATTTCTGTGCGCGATCT SYYGAGTLYG ACGTCTTACTATGGTGCGGGGACCCTA MDVWGQGTTV TACGGTATGGACGTCTGGGGCCAAGGG TVSS ACCACGGTCACCGTCTCCTCAG SEQ ID NO: SEQ ID NO: 575 576 CL-71642 GAGGTGCAGCTGGTGGAGTCTGGGGGA EVOLVESGGG GFTFDDYG INWNGGST AADYYGSGSYYNVP GGTGTGGTACGGCCTGGGGGGTCCCTG VVRPGGSLRL SEQ ID SEQ ID FDY AGACTCTCCTGTGCAGCCTCTGGATTC SCAASGFTFD NO: 582 NO: 583 SEQ ID NO: 584 ACCTTTGATGATTATGGCATGAGCTGG DYGMSWVRQA GTCCGCCAAGCTCCAGGGAAGGGGCTG PGKGLEWVSG GAGTGGGTCTCTGGTATTAATTGGAAT INWNGGSTGY GGTGGTAGCACAGGTTATGCAGACTCT ADSVKGRFTI GTGAAGGGCCGATTCACCATCTCCAGA SRDNAKNSLY GACAACGCCAAGAACTCCCTGTATCTG LOMNSLRAED CAAATGAACAGTCTGAGAGCCGAGGAC TALYYCAADY ACGGCCTTGTATTACTGTGCGGCCGAT YGSGSYYNVP TACTATGGTTCGGGGAGTTATTATAAC FDYWGQGTLV GTCCCCTTTGACTACTGGGGCCAGGGA TVSS ACCCTGGTCACCGTCTCCTCAG SEQ ID NO: SEQ ID NO: 580 581 CL- GAGGTGCAGCTGGTGGAGTCTGGGGGA EVOLVESGGG GFTFDDYG INWIGDNT ARDYFGSGSYYNVP 74570 GGTGTGATACGGCCTGGGGGGTCCCTG VIRPGGSLRL SEQ ID SEQ ID FDY AGACTCTCCTGTGCAGCCTCTGGATTC SCAASGFTFD NO: 582 NO: 587 SEQ ID NO: 588 ACCTTTGATGATTATGGCATGAGCTGG DYGMSWVRçA GTCCGCCAAGCTCCAGGGAAGGGGCTG PGKGLEWVSG GAGTGGGTCTCTGGTATTAATTGGATT INWIGDNTDY GGTGATAACACAGATTATGCAGACTCT ADSVKGRFTI GTGAAGGGCCGATTCACCATCTCCAGA SRDNAKNSLY GACAACGCCAAGAACTCCCTATATCTG LOMNSLRAED CAAATGAACAGTCTGAGAGCCGAGGAC TALYYCARDY ACGGCCTTGTATTACTGTGCGAGAGAT FGSGSYYNVP TACTTTGGTTCGGGGAGTTATTATAAC FDYWGQGTLV GTTCCCTTTGACTACTGGGGCCAGGGA TVSS ACCCTGGTCACCGTCTCCTCAG SEQ ID NO: SEQ ID NO: 585 586

TABLE S5 Sequences of anti-ICOS antibody light chain variable regions obtained from additional clones N terminal E and 5′ nucleotide additions in CL-71642 are shown in bold. These were not recovered in sequencing but were determined to be present in the sequence by comparison against related clones. CDRs are defined according to IMGT. VL_AMINO_ CLONE_ID VL_NUCLEOTIDE_SEQUENCE ACID_SEQ LCDR1 LCDR2 LCDR3 CL-61091 GATATTGTGATGACTCAGTCTCCACTC DIVMTQSPLSL QSLLHS LVS MQALQTPLT TCCCTGCCCGTCACCCCTGGAGAGCCG PVTPGEPASIS NGFNY SEQ ID SEQ ID NO: 593 GCCTCCATCTCCTGCAGGTCTAGTCAG CRSSQSLLHSN SEQ ID NO: 592 AGCCTCCTGCATAGTAATGGATTCAAC GFNYFDWYLQK NO: 591 TATTTCGATTGGTACCTGCAGAAGCCA PGQSPQLLIFL GGACAGTCTCCACAGCTCCTGATCTTT VSNRASGVPDR TTGGTTTCTAATCGGGCCTCCGGGGTC FSGSGSGTDFT CCTGACAGGTTCAGTGGCAGTGGATCA LKISRVEAEDV GGCACAGATTTTACACTGAAAATCAGC GIYYCMQALQT AGAGTGGAGGCTGAGGATGTTGGGATT PLTFGGGTKVE TATTACTGCATGCAAGCTCTACAAACT IK CCGCTCACTTTCGGCGGAGGGACCAAG SEQ ID NO: GTGGAGATCAAAC 590 SEQ ID NO: 589 CL-64536 GATATTGTGATGACTCAGTCTCCACTC DIVMTQSPLSL QSLLHS LGS MQALQTPCS TCCCTGCCCGTCACCCCTGGAGAGCCG PVTPGEPASIS NGYNC SEQ ID SEQ ID NO: 400 GCCTCCATCTCCTGCAGGTCTAGTCAG CRSSQSLLHSN SEQ ID NO 371 AGCCTCCTGCATAGTAATGGATACAAC GYNCLDWYLQK NO: 596 TGTTTGGATTGGTACCTGCAGAAGCCA PGQSPQLLIYL GGGCAGTCTCCACAGCTCCTGATCTAT GSTRASGFPDR TTGGGTTCTACTCGGGCCTCCGGGTTC FSGSGSGTDFT CCTGACAGGTTCAGTGGCAGTGGATCA LKISRVEAEDV GGCACAGATTTTACACTGAAAATCAGC GVYYCMQALQT AGAGTGGAGGCTGAGGATGTTGGGGTT PCSFGQGTKLE TATTACTGCATGCAAGCTCTACAAACT IK CCGTGCAGTTTTGGCCAGGGGACCAAG SEQ ID NO: CTGGAGATCAAAC 595 SEQ ID NO: 594 CL-64837 GATATTGTGATGACTCAGTCTCCACTC DIVMTQSPLSL QSLLHS LGS MQALQTPCS TCCCTGCCCGTCACCCCTGGAGAGCCG PVTPGEPASIS NGYNC SEQ ID SEQ ID NO: 400 GCCTCCATCTCCTGCAGGTCTAGTCAG CRSSQSLLHSN SEQ ID NO: 371 AGCCTCCTGCATAGTAATGGATACAAC GYNCLDWYLQK NO: 596 TGTTTGGATTGGTACCTGCAGAAGCCA PGQSPQLLIYL GGGCAGTCTCCACAGCTCCTGATCTAT GSTRASGFPDR TTGGGTTCTACTCGGGCCTCCGGGTTC FSGSGSGTDFT CCTGACAGGTTCAGTGGCAGTGGATCA LKISRVEAEDV GGCACAGATTTTACACTGAAAATCAGC GVYYCMQALQT AGAGTGGAGGCTGAGGATGTTGGGGTT PCSFGQGTKLE TATTACTGCATGCAAGCTCTACAAACT IK CCGTGCAGTTTTGGCCAGGGGACCAAG SEQ ID NO: CTGGAGATCAAAC 598 SEQ ID NO: 597 CL-64841 GATATTGTGATGACTCAGTCTCCACTC DIVMTQSPLSL QSLLHS LGS MQALQTPCS TCCCTGCCCGTCACCCCTGGAGAGCCG PVTPGEPASIS NGYNC SEQ ID SEQ ID NO: 400 GCCTCCATCTCCTGCAGGTCTAGTCAG CRSSQSLLHSN SEQ ID NO: 371 AGCCTCCTGCATAGTAATGGATACAAC GYNCLDWYLQK NO: 596 TGTTTGGATTGGTACCTGCAGAAGCCA PGQSPQLLIYL GGGCAGTCTCCACAGCTCCTGATCTAT GSTRASGFPDR TTGGGTTCTACTCGGGCCTCCGGGTTC FSGSGSGTDST CCTGACAGGTTCAGTGGCAGTGGATCA LKISRVEAEDV GGCACAGATTCTACACTGAAAATCAGC GVYYCMQALQT AGAGTGGAGGCTGAGGATGTTGGGGTT PCSFGQGTKLE TATTACTGCATGCAAGCTCTACAAACT IK CCGTGCAGTTTTGGCCAGGGGACCAAG SEQ ID NO: CTGGAGATCAAAC 600 SEQ ID NO: 599 CL-64912 GATATTGTGATGACTCAGTCTCCACTC DIVMTQSPLSL QSLLHS LGS MQALQTPCS TCCCTGCCCGTCACCCCTGGAGAGCCG PVTPGEPASIS NGYNC SEQ ID SEQ ID NO: 400 GCCTCCATCTCCTGCAGGTCTAGTCAG CRSSQSLLHSN SEQ ID NO: 371 AGCCTCCTGCATAGTAATGGATACAAC GYNCLDWYLQK NO: 596 TGTTTGGATTGGTACCTGCAGAAGCCA PGQSPQLLIYL GGGCAGTCTCCACAGCTCCTGATCTAT GSTRASGFPDR TTGGGTTCTACTCGGGCCTCCGGGTTC FSGSGSGTDFT CCTGACAGGTTCAGTGGCAGTGGATCA LKI SRVEAEDV GGCACAGATTTTACACTGAAAATCAGC GVYYCMQALQT AGAGTGGAGGCTGAGGATGTTGGGGTT PCSFGQGTKLE TATTACTGCATGCAAGCTCTACAAACT IK CCGTGCAGTTTTGGCCAGGGGACCAAG SEQ ID NO: CTGGAGATCAAAC 602 SEQ ID NO: 601 CL-71642 GAAATTGTGTTGACGCAGTCTCCAGGC EIVLTQSPGTL QSVSSS GAS QQYGSSPFT ACCCTGTCTTTGTCTCCAGGGGAAAGA SLSPGERATLS Y SEQ ID SEQ ID NO: 605 GCCACCCTCTCCTGCAGGGCCAGTCAG CRASQSVSSSY SEQ ID NO: 413 AGTGTTAGCAGCAGCTACTTAGCCTGG LAWYQQKPGQA NO: 426 TACCAGCAGAAACCTGGCCAGGCTCCC PRLLIYGASSR AGGCTCCTCATCTATGGTGCATCCAGC ATGIPDRFSGS AGGGCCACTGGCATCCCAGACAGGTTC GSGTDFTLTIS AGTGGCAGTGGGTCTGGGACAGACTTC RLEPEDFAVYY ACTCTCACCATCAGCAGACTGGAGCCT CQQYGSSPFTF GAAGATTTTGCAGTGTATTACTGTCAG GPGTKVDIK CAGTATGGTAGCTCACCTTTCACTTTC SEQ ID NO: GGCCCTGGGACCAAAGTGGATATCAAA 604 C SEQ ID NO: 603 CL-74570 GAAATTGTGTTGACGCAGTCTCCAGGC EIVLTQSPGTL QSVSSS GAS HQYGNSPFT ACCCTGTCTTTGTCTCCAGGGGAAAGA SLSPGERATLS Y SEQ ID SEQ ID NO: 608 GCCACCCTCTCCTGCAGGGCCAGTCAG CRASQSVSSSY SEQ ID NO: 413 AGTGTTAGCAGCAGCTACTTAGCCTGG LAWYQQKPGQA NO: 426 TACCAGCAGAAACCTGGCCAGGCTCCC PRLLIYGASSR AGGCTCCTCATCTATGGTGCATCCAGC ATGIPDRESGS AGGGCCACTGGCATCCCAGACAGGTTC GSGTDFTLTIS AGTGGCAGTGGGTCTGGGACAGACTTC RLEPEDFAVYY ACTCTCACCATCAGCAGACTGGAACCT CHQYGNSPFTF GAAGATTTTGCAGTATATTACTGTCAC GPGTKVDIK CAGTATGGTAATTCACCATTCACTTTC SEQ ID NO: GGCCCTGGGACCAAAGTGGATATCAAA 607 C SEQ ID NO: 606

Fcab_289 Fc Sequence:

TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

MHEALHNHYTQKSLSLSPGK SEQ ID NO: 620, in which the AB loop (SEQ ID NO: 617), CD loop (SEQ ID NO: 618), and EF loop (SEQ ID NO: 619) are boxed. CH2 domain SEQ ID NO: 613 is double underlined. CH3 domain SEQ ID NO: 616 is single underlined. See also FIG. 2 . STIM001_289 mAb2 Sequences: Variable domain underlined. Leader in bold italic.

Heavy Chain

QVQVVQSGAEVKKPGASVKVSCKASGYTFS TFGITWVRQAPGQGLEWMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTA YMELRSLRSDDTAVYYCARSSGHYYYYGMDVWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDESGYWVSLTCLVK GFYPSDIAVEWESNGEPQYWAKTTPPVLDSDGSFFLYSKLTVSNWRWQL DDFSCSVMHEALHNHYTQKSLSLSPGK HCDR1: GYTFSTFG HCDR2: ISAYNGDT HCDR3: ARSSGHYYYYGMDV

CAGGTTCAGGTGGTGCAGTCTGGAGCTGAGG TGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTA CACCTTTTCCACCTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAA GGGCTTGAATGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACT ATGCACAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCCAC GAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCC GTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGG ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAA GGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGC GGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTG TGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTT CCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTG ACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGA ACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTC CTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTG GGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGA TGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCA CGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTG CACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACC GGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAA AGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAA AAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACA CACTGCCCCCTAGCAGGGACGAGAGCGGGTACTGGGTGTCCCTGACCTG TCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCC AACGGCGAGCCTCAGTACTGGGCCAAGACCACCCCCCCTGTGCTGGACT CCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGAGCAACTGGCG GTGGCAGCTGGACGACTTCTCCTGCTCCGTGATGCACGAGGCCCTGCAC AACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG Light Chain

DIVMTQSPLSLPVTPGEPASISCRSSQSL LHSNEYNYLDWYLQKPGQSPQLLIFLGSNRASGVPDRFSGSGSGTDFTL KITRVEAEDVGIYYCMQSLQTPLTFGGGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC LCDR1: QSLLHSNEYNY LCDR2: LGS LCDR3: MQSLQTPLT

GATATTGTGATGACTCAGTCTCCACTCTC CCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGT CAGAGCCTCCTGCATAGTAATGAATACAACTATTTGGATTGGTACCTGC AGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCG GGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGAT TTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATT ACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAA GGTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCA CCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGC TGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAA CGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCC AAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCG ACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCT GTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT STIM003_289 mAb2 Sequences: Variable domain underlined. Leader in bold italic.

Heavy Chain

EVQLVESGGGVVRPGGSLRLSCVASGVTF DDYGMSWVRQAPGKGLEWVSGINWNGGDTDYSDSVKGRFTISRDNAKN SLYLQMNSLRAEDTALYYCARDFYGSGSYYHVPFDYWGQGILVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDES GYWVSLTCLVKGFYPSDIAVEWESNGEPQYWAKTTPPVLDSDGSFFLY SKLTVSNWRWQLDDFSCSVMHEALHNHYTQKSLSLSPGK HCDR1: GVTFDDYG HCDR2: INWNGGDT HCDR3: ARDFYGSGSYYHVPFDY Heavy chain aa sequence without leader SEQ ID NO: 621 Heavy chain aa sequence including leader SEQ ID NO: 622 Heavy chain encoding nucleic acid SEC) ID NO: 612.

GAGGTGCAGCTGGTGGAGTCTGGGGGAGG TGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGA GTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGA AGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGA TTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC AAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGG CCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCA CGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA GCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGT CCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTT CCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGA GTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGT CCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACAT CTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTG GAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCC CTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAA GGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTG GATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACG GCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAA CTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG CTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTG CCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACC CCAGGTGTACACACTGCCCCCTAGCAGGGACGAGAGCGGGTACTGGGTG TCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGG AATGGGAGTCCAACGGCGAGCCTCAGTACTGGGCCAAGACCACCCCCCC TGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTG AGCAACTGGCGGTGGCAGCTGGACGACTTCTCCTGCTCCGTGATGCACG AGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGG CAAG

Light Chain

EIVLTQSPGTLSLSPGERATLSCRASQSV SRSYLAWYQQKRGQAPRLLIYGASSRATGIPDRFSGDGSGTDFTLSISR LEPEDFAVYYCHQYDMSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC LCDR1: QSVSRSY LCDR2: GAS LCDR3: HQYDMSPFT Light chain aa sequence without leader SEQ ID NO: 623 Light chain aa sequence with leader SEQ ID NO: 624 Light chain encoding nucleic acid SEQ ID NO: 614.

GAAATTGTGTTGACGCAGTCTCCAGGGACCC TGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCA GAGTGTTAGCAGAAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAG GCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCC CAGACAGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCAT CAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTAT GATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC GTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCA GCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTAC CCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCG GCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTA CTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCAC AAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGA CCAAGTCTTTCAACCGGGGCGAGTGT

REFERENCE

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1-40. (canceled)
 41. A composition comprising isolated nucleic acid sequences or a set of nucleic acid sequences encoding a multispecific antibody comprising a Fab and an Fc region, wherein the Fab comprises an ICOS binding site and wherein the Fc region comprises a PD-L1 binding site, wherein the PD-L1 binding site is provided by a CH3 domain in which residues 14 to 18 are substituted by SGYW (SEQ ID NO: 617), residues 45.1 to 78 are substituted by EPQYWA (SEQ ID NO: 618), and residues 92 to 101 are substituted by SNWRWQLDD (SEQ ID NO: 619).
 42. A method of treating cancer in a human patient comprising administering a multispecific antibody comprising a Fab and an Fc region, wherein the Fab comprises an ICOS binding site and wherein the Fc region comprises a PD-L1 binding site, wherein the PD-L1 binding site is provided by a CH3 domain in which residues 14 to 18 are substituted by SGYW (SEQ ID NO: 617), residues 45.1 to 78 are substituted by EPQYWA (SEQ ID NO: 618), and residues 92 to 101 are substituted by SNWRWQLDD (SEQ ID NO: 619).
 43. (canceled)
 44. (canceled)
 45. The method of claim 42, wherein the cancer is associated with Tregs and/or tests positive for expression of ICOS and FOXP3.
 46. The method of claim 42, wherein the cancer is head and neck cancer, non small cell lung cancer or cervical cancer.
 47. The method of claim 42, wherein the method further comprises administering a CTLA-4 antagonist or an anti-CTLA-4 antibody to the patient.
 48. The method of claim 47, wherein the anti-CTLA-4 antibody is ipilimumab or tremelimumab.
 49. The method of claim 42, wherein the method further comprises administering a PD-1 antagonist or an anti-PD-1 antibody to the patient.
 50. The method of claim 49, wherein the anti-PD-1 antibody is pembrolizumab, nivolumab or genolimzumab.
 51. The nucleic acid sequences or set of nucleic acid sequences of claim 41, wherein the Fab comprises a VH domain having a HCDR1, HCDR2 and HCDR3 sequences wherein HCDR1 is SEQ ID NO: 405, HCDR2 is SEQ ID NO: 406, and HCDR3 is SEQ ID NO:
 407. 52. The nucleic acid sequences or set of nucleic acid sequences of claim 41, wherein the Fab comprises a VL domain having LCDR1, LCDR2 and LCDR3 sequences wherein LCDR1 is SEQ ID NO: 412, LCDR2 is SEQ ID NO: 413, and LCDR3 is the SEQ ID NO
 414. 53. The nucleic acid sequences or set of nucleic acid sequences of claim 41, wherein the Fab comprises a VH domain amino acid sequence at least 90% identical to SEQ ID NO: 408 and a VL domain amino acid sequence at least 90% identical to SEQ ID NO:
 415. 54. A vector comprising the nucleic acid sequences or set of nucleic acid sequences of claim
 41. 55. An isolated recombinant host cell transformed with the nucleic acid sequences or set of nucleic acid sequences of claim
 54. 56. A method of making the multispecific antibody comprising culturing the host cell of claim 55 under conditions to produce the antibody and recovering the antibody.
 57. A nucleic acid sequence or a set of nucleic acid sequences encoding a multispecific antibody comprising a Fab and an Fc region, wherein the Fab comprises an ICOS binding site wherein the Fc region comprises a PD-L1 binding site, wherein the PD-L1 binding site is provided by a CH3 domain in which residues 14 to 18 are substituted by SGYW (SEQ ID NO: 617), residues 45.1 to 78 are substituted by EPQYWA (SEQ ID NO: 618), and residues 92 to 101 are substituted by SNWRWQLDD (SEQ ID NO: 619), and wherein the multispecific antibody comprises a heavy chain comprising amino acid sequence SEQ ID NO:
 621. 58. The nucleic acid sequence or a set of nucleic acid sequences of claim 57, comprising a light chain comprising amino acid sequence SEQ ID NO:
 623. 59. The nucleic acid sequence or a set of nucleic acid sequences of claim 57, comprising two heavy-light chain pairs, in which the heavy chain comprises amino acid sequence SEQ ID NO: 621 and the light chain comprises amino acid sequence SEQ ID NO:
 623. 60. A vector comprising the nucleic acid sequence or a set of nucleic acid sequences of claim
 57. 61. An isolated recombinant host cell transformed with the nucleic acid sequence or a set of nucleic acid sequences of claim
 57. 62. A method of making the multispecific antibody comprising culturing the host cell of claim 61 under conditions to produce the antibody and recovering the antibody. 